Desethylhydroxychloroquine for the treatment of diseases associated with inflammation

ABSTRACT

Compositions and methods are provided for inhibiting or treating the early and established stages of inflammatory diseases by administration of an effective dose of the desethylhydroxychloroquine (DHCQ). A benefit of the methods is the ability to deliver a dose of agent that is effective in treating inflammation while sparing the individual from retinal toxicity.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 15/446,860 filed Mar. 1, 2017, which is a Continuation ofapplication Ser. No. 14/214,307 filed Mar. 14, 2014, now U.S. Pat. No.9,616,057 issued Apr. 11, 2017, which claims benefit of U.S. ProvisionalPatent Application No. 61/791,320, filed Mar. 15, 2013, whichapplications are incorporated herein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract A1069160and HV000242 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

In various embodiments, the present invention relates to compositionscomprising desethylhydroxychloroquine (DHCQ) and methods for preventingand treating inflammatory diseases or diseases associated withinflammation with such compositions. Inflammatory diseases, or diseasesassociated with inflammation, include autoimmune diseases, degenerativediseases, metabolic diseases, cardiovascular diseases, chronicinfections, and malignancies. In other embodiments, the presentinvention relates to the prevention of or treatment of such inflammatorydiseases or diseases associated with inflammation, including preventingthe onset of disease in individuals at increased risk for developing thedisease, preventing the progression of disease in individuals at earlystages of the disease, and treating established disease.

BACKGROUND OF THE INVENTION

Inflammation contributes to pathogenesis of inflammatory diseases, ordiseases associated with inflammation, such as autoimmune diseasesincluding rheumatoid arthritis (RA), systemic lupus erythematosus (SLE),multiple sclerosis (MS), and autoimmune hepatitis; degenerative diseaseswith an inflammatory component such as osteoarthritis (OA), Alzheimer'sdisease (AD), and macular degeneration; chronic infections such as HIV;metabolic diseases with an inflammatory component such as type IIdiabetes, metabolic syndrome, and atherosclerosis; and malignantdiseases including cancers. Aminoquinolines including hydroxychloroquine(HCQ) are used as anti-inflammatory agents to treat certain inflammatorydiseases.

Aminoquinolines are derivatives of quinoline that are most notable fortheir roles as antimalarial drugs, but they also possessanti-inflammatory properties. Examples of drugs of the aminoquinolineclass include, but are not limited to, 4-aminoquinolines, such asamodiaquine, hydroxychloroquine (HCQ), chloroquine; and8-aminoquinolines, such as primaquine and pamaquine. 4-Aminoquinoline isa form of aminoquinoline with the amino group at the 4-position of thequinoline. A variety of derivatives of 4-aminoquinoline are antimalarialagents, and examples include amodiaquine, chloroquine, and HCQ.

The 4-aminoquinoline HCQ was initially developed as hydroxychloroquinesulfate (HCQ sulfate) for use as an antimalarial drug.Hydroxychloroquine sulfate is sold under the trade names Plaquenil™,Axemal™ (in India), Dolquine™, and Quensyl™, and is also widely used toreduce inflammation in the treatment of systemic lupus erythematosus,rheumatoid arthritis, Sjögren's Syndrome, and porphyria cutanea tarda.HCQ differs from chloroquine by having a hydroxyl group at the end ofthe side chain: The N-ethyl substituent is beta-hydroxylated. It isavailable for oral administration as hydroxychloroquine sulfate(Plaquenil), of which 200 mg contains 155 mg hydroxychloroquine base inchiral form. In addition to 155 mg of hydroxychloroquine base, eachPlaquenil tablet contains the following inactive ingredients: sulfate(SO₄), anhydrous lactose, croscarmellose sodium, glyceryl triacetate,hypromellose, magnesium stearate, microcrystalline cellulose,polydextrose, polyethylene glycol, povidone, sodium lauryl sulfate andtitanium dioxide. Hydroxychloroquine sulfate has similarpharmacokinetics to chloroquine phosphate, being quickly absorbed by thegastrointestinal tract and eliminated by the kidney. Cytochrome P450enzymes (CYP 2D6, 2C8, 3A4, and 3A5)N-desethylate HCQ toN-desethylhydroxychloroquine (Kalia et al. (2007) Dermatologic Therapy20 (4): 160-174).

The most common adverse effects of HCQ therapy are mild nausea andoccasional stomach cramps with mild diarrhea. The most serious adverseeffects affect the eye. One of the most serious side effects of chronicHCQ use is ocular and retinal toxicity (Flach. Transactions of theAmerican Ophthalmological Society, 2007, 105: 191-4; discussion 195-7).

Prolonged use of HCQ, chloroquine, or other aminoquinolines isassociated with the development of eye toxicity (Marmor et al. ArthritisCare Res. 2010; 62(6):775-84; Levy et al, Incidence ofhydroxychloroquine retinopathy in 1,207 patients in a large multicenteroutpatient practice. Arthritis Rheumatism 1997, 40(8):1482-6; Mavrikakiset al, The incidence of irreversible retinal toxicity in patientstreated with hydroxychloroquine: a reappraisal. Opthalmology, 2003,110(7):1321-6). The incidence of such toxicity increases markedly withthe duration of therapy, with ophthalmoscopically visualized loss ofretinal pigmented epithelium in approximately 0.5-1% of HCQ sulfatetreated humans after 5 years; and approximately 2% of HCQ sulfatetreated humans after 10-15 years. Considerably higher rates of toxicityare observed with chloroquine. Notably, despite the observed rates ofretinal toxicity, total rates of physician discontinuation of HCQ forearlier eye problems (including asymptomatic changes noted onophthalmologic examination) approach 7% of HCQ sulfate treated patientsover 5 years (Marmor et al. Rates and predictors of hydroxychloroquineretinal toxicity in patients with rheumatoid arthritis and systemiclupus erythematosus, Arthritis Care Res. 2010; 62(6):775-84).

Toxicity due to HCQ may occur in two distinct areas of the eye: thecornea and the macula. The cornea may become affected (relativelycommonly) by an innocuous vortex keratopathy that is characterized bywhorl-like corneal epithelial deposits. Changes to the macula (acomponent of the retina) are more serious and are related to dosage andduration of HCQ use. Advanced retinopathy is characterized by reductionof visual acuity and a “bull's-eye” macular lesion, which is absent inthe earlier stages. Bull's eye maculopathy and/or paracentral scotomaare clinical features of HCQ retinopathy.

Macular retinal toxicity is related to the total cumulative dose. Peopletaking 400 mg of HCQ S04 or less per day generally have lower risk ofmacular retinal toxicity, and the risk increases when a person takes themedication for more than 5 years or takes a cumulative dose of more than1000 grams, and at a dose of 400 mg/day of HCQ sulfate the cumulativedose of 1000 grams is reached at 7 years of dosing (400 mg/day HCQsulfate×365 days/year×7 years=1022 grams of HCQ sulfate) (Wolfe andMarmor, Rates and Predictors of Hydroxychloroquine Retinal Toxicity inPatients with Rheumatoid Arthritis and Systemic Lupus Erythematosus,Arthritis Care and Research, 2010, 62(6):775-784; Marmor et al. (2011)Ophthalmology 118 (2): 415-22). The risk of retinal toxicity was foundto be 5 times higher after 7 years of usage or 1000 grams of totalexposure (Marmor et al. Arthritis Care Res. 2010; 62(6):775-84). Regulareye screening, even in the absence of visual symptoms, is recommended tobegin when either of these risk factors is present (Marmor et al. (2011)Ophthalmology 118 (2): 415-22). In a study of 3,995 patients withrheumatoid arthritis or systemic lupus erythematosus who were treatedwith HCQ sulfate, eye examinations to monitor for HCQ toxicity wereobtained annually in 50.5% of patients and every 6 months in 40.4% ofpatients (Marmor et al. Arthritis Care Res. 2010; 62(6):775-84).

The exact mechanisms underlying HCQ-induced retinal toxicity, includingretinal macular toxicity, are not clear. Studies to date have identifiedretinal accumulation of HCQ to levels much higher than those observed inother tissues and in the blood. In addition, HCQ binds to melanin in theretinal pigment epithelium (RPE), and such binding may contribute to orprolong HCQ's toxic effects. Some studies have demonstrated that bothchloroquine and HCQ are associated with increased lipofuscin formation,a process known to be accelerated by increased lysosomal pH andintra-lysosomal oxidation during degradation of auto-/heterophagocytosedmaterial (Sundelin et al. APMIS [Acta Pathologica, MicrobiologicaImmunologica Scandinavica]. 2002; 110(6):481-9). Findings suggest thatchloroquine blocks attachment of autophagosomes to lysosomes, therebyresulting in the accumulation of lipofuscin and persistence chloroquineand other aminoquinolines in the retinal pigmented epithelial cells(Yoon et al, Induction of lysosomal dilatation, arrested autophagy, andcell death by chloroquine in cultured ARPE-19 cells, Invest OphthalmolVis Sci. 2010, 51(11):6030-7). Additionally, because melanin within theRPE has a role in neutralizing oxidative free radicals, it has beensuggested that the presence of excessive levels of such free radicalsmay contribute to the pathogenesis of HCQ-induced retinal toxicity(Sundelin et al. APMIS. 2002; 110(6):481-9).

Retinal toxicity induced by chloroquine and HCQ is characterized by afine mottling of the macula, arteriolar narrowing, peripheral retinalpigmentation, loss of the foveal reflex and, in advanced cases, by adepigmented macula surrounded by a pigmented ring, a finding termed“bull's-eye maculopathy” (Mecklenburg et al, Toxicol Pathol. 2007;35(2):252-67). In the early stages of retinal toxicity, patients maynotice decreased visual acuity, blurred vision, decreased color andnight vision, as well as a paracentral scotoma (Mecklenburg et al,Toxicol Pathol. 2007; 35(2):252-67). HCQ retinopathy is related to thetotal cumulative dose and develops slowly, but can progress to a moreserious loss of central and peripheral vision for which there is noknown treatment (Marmor et al., Arthritis Care Res. 2010; 62(6):775-84).

Current recommendations for screening for chloroquine and HCQretinopathy are contained in the “2011 AAO Revised Recommendations”described in Marmor et al. (Revised recommendations on screening forchloroquine and hydroxychloroquine retinopathy, Ophthalmology. 2011,118(2):415-22). The recommendations include performing a baselineexamination within the first year of patients startinghydroxychloroquine or chloroquine therapy to serve as a reference pointand to rule out pre-existing maculopathy, which frequentlycontraindicates use of these drugs. Annual screening for eye toxicity isrecommended to begin after 5 years, or sooner if there are additionalrisk factors including cumulative dose >1000 grams of HCQ sulfate, useof a daily dose of HCQ sulfate >400 mg/day (or >6.5 mg/kg HCQ sulfatefor lean body weight for short individuals), advanced age, kidney orliver dysfunction, or retinal disease or maculopathy (Marmor et al,Opthamology. 2011, 118(2):415-22).

As described in the “2011 AAO Revised Recommendations” (Revisedrecommendations on screening for chloroquine and hydroxychloroquineretinopathy, Ophthalmology. 2011, 118(2):415-22), newer objective tests,such as multifocal electroretinogram (mfERG), spectral domain opticalcoherence tomography (SD-OCT), and fundus autofluorescence (FAF), can bemore sensitive than visual field tests. It is now recommended that alongwith white 10-2 automated field threshold tests, at least one of theseprocedures be used for routine screening when available. When fieldtests are performed independently, even the most subtle 10-2 fieldchanges should be taken seriously and are an indication for evaluationby objective testing. Because mfERG testing is an objective test thatevaluates function, it may be used in place of visual field tests.Amsler grid testing is no longer recommended. Fundus examinations areadvised for documentation, but visible bull's-eye maculopathy is a latechange, and the goal of screening is to detect toxicity at an earlierstage. Further, patients should be aware of the risk of toxicity and therationale for screening (to detect early changes and minimize visualloss, not necessarily to prevent it). The drugs should be stopped ifpossible when toxicity is detected or strongly suspected (Marmor et al,Ophthalmology. 2011, 118(2):415-22).

Although HCQ and other aminoquinolines have been used to treatinflammatory diseases, as discussed herein, prolonged administration ofHCQ and other aminoquinolines is associated with deposition of theaminoquinoline in the retina and the development of retinal toxicity.Accordingly, there is a need for improved pharmaceutical compositionswhich provide anti-inflammatory activity while exhibiting less retinalaccumulation and less retinal toxicity as compared to HCQ and otheraminoquinolines.

SUMMARY OF THE INVENTION

In various embodiments, compositions and methods are provided forpreventing or treating inflammatory diseases or diseases associated withinflammation. In particular, the compositions and methods of the presentinvention are suitable for treating various patient populations, and atvarious stages in the disease process, including in the at-risk period,early stages and established inflammatory diseases, including autoimmunediseases, degenerative inflammatory diseases, metabolic inflammatorydiseases, and other inflammatory diseases and diseases associated withinflammation by administration to an individual an effective dose of theaminoquinoline desethylhydroxychloroquine (DHCQ). Treatment ofinflammatory disease at an early time point by the compositions andmethods of the invention can substantially reduce or prevent diseasedevelopment, clinical symptoms, or disease progression. In someembodiments treatment is initiated when individuals are at increasedrisk for development of a disease to prevent development of the disease,to treat early signs or symptoms of disease, or to reverse early signsor symptoms of disease. Individuals who are at increased risk fordevelopment of disease may be asymptomatic, may be asymptomatic but haveearly signs of disease, or may have early symptoms of disease. In otherembodiments, treatment is initiated when individuals have early-stagedisease to prevent progression of disease, to treat early signs orsymptoms of disease, or to reverse early signs or symptoms of disease.When individuals have early-stage disease they have early symptoms orsigns of disease, may have intermittent or mild symptoms, or may beasymptomatic and only exhibit signs of disease. In other embodiments,treatment is initiated when individuals have established disease toprevent progression of disease, to treat signs and symptoms of disease,or to reverse signs and symptoms of disease. Administration of thepharmaceutical composition of this invention may continue for anextended period of time, for example over a period of months or years.In some embodiments, treatment with DHCQ is continued for at least aboutone year. In particular embodiments, treatment with DHCQ is continuedfor at least about 1 year. In some embodiments, treatment with DHCQ iscontinued for at least about 7 years. In some embodiments, treatmentwith DHCQ is continued for at least about 5 years. In some embodiments,treatment with DHCQ is continued for at least about 10 years. In someembodiments, treatment with DHCQ is continued for at least about 15years. In some embodiments, treatment with DHCQ is continued for atleast about 20 years. In some embodiments, treatment with DHCQ iscontinued for the lifetime of the individual.

In some embodiments, a pharmaceutical composition comprising aneffective dose of desethylhydroxychloroquine, e.g. DHCQ, and apharmaceutically acceptable excipient is provided. In some embodiments,the pharmaceutical formulation comprises, consists, or consistsessentially of DHCQ or an equivalent in a daily dose of at least about50 mg per day (about 0.83 mg/kg/day), about 100 mg per day (about 1.66mg/kg/day), about 155 mg per day (about 2.58 mg/kg/day), about 200 mgper day (about 3.33 mg/kg/day), about 250 mg per day (about 4.16mg/kg/day), about 300 mg per day (about 5 mg/kg/day), about 310 mg perday (about 5.16 mg/kg/day), about 350 mg per day (about 5.83 mg/kg/day),about 00 mg per day (about 6.67 mg/kg/day), about 450 mg per day (about7.5 mg/kg/day), about 465 mg per day (about 7.75 mg/kg/day), about 500mg per day (about 8.33 mg/kg/day), about 550 mg per day (about 9.16mg/kg/d), about 600 mg per day (about 10 mg/kg/day), about 620 mg perday (about 10.3 mg/kg/day), about 800 mg per day (about 13.3 mg/kg/day),about 930 mg/kg/day (about 15.5 mg/kg/day), about 1000 mg per day (about16.67 mg/kg/day), about 1200 mg per day (about 20 mg/kg/day), about 1300mg per day (about 21.67 mg/kg/day), about 1400 mg per day (about 23.3mg/kg/day), about 1500 mg per day (about 25 mg/kg/day), or about 1600 mgper day (about 26.67 mg/kg/day).

In some embodiments, the pharmaceutical formulation comprises, consists,or consists essentially of DHCQ or an equivalent in a daily dose fromabout 50 mg per day (about 0.83 mg/kg/day) to about 200 mg per day(about 3.33 mg/kg/day), about 100 mg per day (about 1.67 mg/kg/day) toabout 400 mg per day (about 6.67 mg/kg/day), about 200 mg/per day (about3.33 mg/kg/day) to about 450 mg per day (about 7.5 mg/kg/day), fromabout 300 mg per day (about 5 mg/kg/day) to about 550 mg per day (about9.16 mg/kg/day), from about 400 mg per day (about 6.67 mg/kg/day) toabout 600 mg per day (about 10 mg/kg/day), from about 500 mg per day(about 8.33 mg/kg/day) to about 700 mg per day (about 11.67 mg/kg/day),from about 550 mg per day (about 9.16 mg/kg/day) to about 750 mg per day(about 12.5 mg/kg/day), from about 600 mg per day (about 10 mg/kg/day)to about 800 mg per day (about 13.3 mg/kg/day), from about 700 mg perday (about 11.67 mg/kg/day) to about 1000 mg per day (about 16.67mg/kg/day), from about 800 mg per day (about 13.3 mg/kg/day) to about1600 mg per day (about 26.67 mg/kg/day), from about 900 mg per day(about 15 mg/kg/day) to about 1200 mg per day (about 20 mg/kg/day), fromabout 1200 mg per day (about 15 mg/kg/day) to about 1600 mg per day(about 26.67 mg/kg/day), from about 1400 mg per day (about 23.3mg/kg/day) to about 1600 mg per day (about 26.67 mg/kg/day).

The mg/kg/day dosage used herein and throughout this document is basedon an ideal body weight of humans of 60 kg (Marmor et al, RevisedRecommendations on Screening for Chloroquine and HydroxychloroquineRetinopathy, Opthalmology 2011, 118:415-422; Pai et al, The Origin ofthe “Ideal” Body Weight Equations. The Annals of Pharmacotherapy, 2000,34 (9): 1066-1069; Walpole et al, BMC Public Health (BMC Public Health2012, 12:439) 12: 439). In other embodiments, the mg/kg/day dosage isbased on an average lean body weight of humans of 60 kg (Mackenzie A H.Dose refinements in long-term therapy of rheumatoid arthritis withantimalarials. Am J Med. 1983, 75(suppl):40-45; Hume, R. Prediction oflean body mass from height and weight. Journal of clinical pathology,1966, 19 (4): 389-91. PMC 473290. PMID 5929341; Wolfe and Marmor, Ratesand Predictors of Hydroxychloroquine Retinal Toxicity in Patients withRheumatoid Arthritis and Systemic Lupus Erythematosus, Arthritis Careand Research, 2010, 62(6):775-784).

Depending on the patient and condition being treated and on theadministration route, the DHCQ will generally be administered in dosagesof about 50-1600 mg per day (0.83-26.6 mg/kg/g/day). The range is broadsince in general the efficacy of a therapeutic effect for differentmammals varies widely with doses typically being 20, 30, 40 or even 50times smaller (per unit body weight) in humans than in the mouse.Similarly the mode of administration can have a large effect on dosage.Thus for example oral dosages in the mouse may be ten times theinjection dose in the mouse. As a result, the preferred range for miceis about 2.5 to 150 mg/kg/day, and in some embodiments in mice about 10to 100 mg/kg/day, while for humans it may be about 0.83-26.6 mg/kg/day,and in some embodiments about 6.67-13.3 mg/kg/day. A typical adult humandosage may be about 400 mg per day, or about 500 mg per day, or about550 mg per day, or about 600 mg per day, or about 700 mg per day, orabout 750 mg per day, or about 800 mg per day, or the amounts describedin the daily dosages elsewhere in this application. The total daily dosemay be taken in divided two times per day, or three times per day doses.In one embodiment, about 200 mg twice per day (about 400 mg total perday, about 6.67 mg/kg/day), or about 250 mg twice per day (about 500 mgtotal per day, about 8.33 mg/kg/day), or about 275 mg twice per day(about 550 mg total per day, about 9.16 mg/kg/day), or about 300 mgtwice per day (about 600 mg total per day, about 10 mg/kg/day), or about350 mg twice per day (about 700 mg total per day, about 11.66 mg/kg/day)or about 400 mg twice per day (about 800 mg total per day, about 13.3mg/kg/day), or in the total dosages described elsewhere in thisapplication. Doses may be taken with the meals.

In some embodiments, treatment may comprise administering a synergisticcombination of desethylhydroxychloroquine (DHCQ) with one or more activeagents, wherein the synergistic combination comprises, consists, orconsists essentially of DHCQ and one or more statins, e.g. atorvastatin,fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin,rosuvastatin, simvastatin, etc. The active agents of the synergisticcombination can be administered separately, or can be co-formulated in asingle unit dose (i.e., a “fixed dose combination”). Each or all of theactive agents can be formulated in various ways, including withoutlimitation a solid oral dosage form.

In some embodiments a package suitable for use in commerce is providedfor treating inflammation according to the methods of the invention,e.g. a pharmaceutical formulation comprising, consisting of, orconsisting essentially of DHCQ, optionally in combination with one ormore additional agents, e.g. one or more statins as described herein;and associated with said package (e.g., a carton or container), printedinstructional and informational material, which may be attached to saidcarton or to said container enclosed in said carton, or displayed as anintegral part of said carton or container, said instructional andinformational material stating in words which convey to a reader thereofthat the active ingredients, when administered to an individual in theearly stages of inflammatory disease, will ameliorate, diminish,actively treat, reverse or prevent any injury, damage or loss of tissuesubsequent to early stages of disease. The package comprising a cartonand/or container as described herein may conform to all regulatoryrequirements relating to the sale and use of drugs, including especiallyinstructional and informational material.

In some embodiments the methods of the invention comprise the step ofidentifying individuals “at-risk” for development of, or in the“early-stages” of, an inflammatory disease. “At risk” for development ofan inflammatory disease includes: (1) individuals whom are at increasedrisk for development of an inflammatory disease, and (2) individualsexhibiting a “pre-clinical” disease state, but do not meet thediagnostic criteria for the inflammatory disease (and thus are notformally considered to have the inflammatory disease).

Individuals “at increased risk” for development (also termed “at-risk”for development) of an inflammatory disease are individuals with ahigher likelihood of developing an inflammatory disease or diseaseassociated with inflammation compared to the general population. Suchindividuals can be identified based on their exhibiting or possessingone or more of the following: a family history of inflammatory disease;the presence of certain genetic variants (genes) or combinations ofgenetic variants which predispose the individual to such an inflammatorydisease; the presence of physical findings, laboratory test results,imaging findings, marker test results (also termed “biomarker” testresults) associated with development of the inflammatory disease, ormarker test results associated with development of a metabolic disease;the presence of clinical signs related to the inflammatory disease; thepresence of certain symptoms related to the inflammatory disease(although the individual is frequently asymptomatic); the presence ofmarkers (also termed “biomarkers”) of inflammation; and other findingsthat indicate an individual has an increased likelihood over the courseof their lifetime to develop an inflammatory disease or diseaseassociated with inflammation. Most individuals at increased risk fordevelopment of an inflammatory disease or disease associated withinflammation are asymptomatic, and are not experiencing any symptomsrelated to the disease that they are at an increased risk fordeveloping.

Included, without limitation, in the group of individuals at increasedrisk of developing an inflammatory disease or a disease associated withinflammation, are individuals exhibiting “a pre-clinical disease state”.The pre-disease state may be diagnosed based on developing symptoms,physical findings, laboratory test results, imaging results, and otherfindings that result in the individual meeting the diagnostic criteriafor the inflammatory disease, and thus being formally diagnosed.Individuals with “pre-clinical disease” exhibit findings that suggestthat the individual is in the process of developing the inflammatorydisease, but do not exhibit findings, including the symptoms, clinicalfindings, laboratory findings, and/or imaging findings, etc. that arenecessary to meet the diagnostic criteria for a formal diagnosis of theinflammatory disease. In some embodiments, individuals exhibiting apre-clinical disease state possess a genetic variant or a combination ofgenetic variants that place them at increased risk for development ofdisease as compared to individuals who do not possess that geneticvariant or that combination of genetic variants. In some embodiments,these individuals have laboratory results, or physical findings, orsymptoms, or imaging findings that place them at increased risk fordevelopment of an inflammatory disease. In some embodiments, individualswith preclinical disease states are asymptomatic. In some embodiments,individuals with pre-clinical disease states exhibit increased ordecreased levels of the expression of certain genes, certain proteins,inflammatory markers, metabolic markers, and other markers.

In some embodiments, individuals at increased risk for an inflammatorydisease exhibit increased markers of inflammation (also termed“inflammatory markers” or “inflammatory biomarkers”). Examples ofmolecular markers of inflammation include c-reactive protein (CRP),high-sensitivity CRP (hs-CRP) (or regular CRP), erythrocytesedimentation rate (ESR), serum amyloid A, serum amyloid P, fibrinogen,cytokines in blood or other biological fluids, a cytokine, an antibody(such as an autoantibody, or an anti-microbial antibody), a DNAsequence, a RNA sequence (for example, mRNA encoding one or morecytokines or other immune molecules), other markers of inflammation, orcombinations thereof. The method can include determining the presence ofinflammation prior to treatment, for example by detection and analysisof one or more markers of inflammation, where an individual in an earlystage of disease showing signs of inflammation is selected for treatmentwith a formulation of the invention. In some embodiments the treatmentameliorates, diminishes, actively treats, reverses or prevents tissueinjury. In some embodiments the inflammatory disease is an autoimmunedisease, for example RA, multiple sclerosis, systemic lupuserythematosus, Sjogren's Syndrome, etc. In some embodiments, the markerof inflammation (or inflammatory marker) is a metabolic marker (alsotermed herein as a “metabolic disease marker”). In some embodiments thedisease comprises an inflammatory component contributing to a metabolicdisease, for example metabolic syndrome, type II diabetes, insulinresistance, atherosclerosis, etc. In some embodiments the disease is adegenerative disease such as OA, Alzheimer's disease, or maculardegeneration.

In some embodiments the marker of inflammation is an abnormal metabolicmarker (also termed herein as a “metabolic disease marker”), and theabnormal metabolic marker is selected from the group consisting of ablood pressure of about 140/90 mmHg or more, plasma triglyceride levelsof about 1.7 mmol/L or more, high-density lipoprotein cholesterol(HDL-C) levels of about 0.9 mmol/L or less for males and about 1.0mmol/L or less for females, a microalbuminuria:urinary albumin excretionratio of about 20 μg/min or more, or an albumin:creatinine ratio ofabout 30 mg/g or more, a fasting blood glucose greater than about 100mg/dL, 2 hour post-prandial blood glucose greater than about 200, or ahemoglobin A1c greater than about 6.5 mg/dL. Patients with abnormalmetabolic markers are at increased risk for developing an inflammatorydisease or disease associated with inflammation.

In some embodiments the methods of the invention comprise the step ofidentifying and treating individuals at increased risk for developmentof an inflammatory disease or disease associated with inflammation.These individuals at increased risk for development of an inflammatorydisease can have risk factors for disease and/or be in a “pre-clinical”state as described herein, and are sometimes asymptomatic. Treatmentwith DHCQ alone or synergistic combinations thereof with a statin, asdescribed herein, at this point is exceptionally valuable in preventingdevelopment of the inflammatory disease or disease associated withinflammation; however, it is important to prescribe a safe andefficacious therapy that can be tolerated over long periods of time, asis provided by the present invention.

The determination of “early-stage disease” in an individual can compriseanalyzing the individual for the presence of at least one markerindicative of the presence of early disease. In some embodiments themethod comprises analyzing an individual for the presence of one, two,three, four, or more markers that are predictive for an individual beingat increased risk for developing or in the early-stages of aninflammatory disease or disease associated with inflammation. In someembodiments at least one of the marker(s) is an imaging marker,including without limitation: arthroscopy, radiographic imaging,ultrasound imaging, magnetic resonance imaging (MRI), computedtomography (CT), etc. In some embodiments at least one of the marker(s)is a molecular marker of inflammation, where a biological sample isobtained from the individual and analyzed for the presence of amolecule, e.g. high-sensitivity C-reactive protein (or regular CRP),erythrocyte sedimentation rate, serum amyloid A, serum amyloid P,fibrinogen, a cytokine, antibody (autoantibody or anti-microbialantibody), cartilage component, protease, RNA molecule (for example,mRNA encoding one or more cytokines or other immune molecules), etc. andcompared to a control or reference value, wherein altered level of themolecular marker is indicative of early disease. In some embodiments,early-stage disease is defined by the presence of symptoms for less thanabout 6 months. In some embodiments, early-stage disease is defined bybeing formally diagnosed with the inflammatory disease for less thanabout 6 months. In some embodiments, early-stage disease is associatedwith no symptoms. In some embodiments, early-stage disease is associatedwith mild symptoms. In some embodiments, early-stage disease isassociated with intermittent symptoms, such as symptoms occurring onlyonce every couple years, or symptoms occurring once every couple months,or symptoms occurring once every couple days, or symptoms occurring foronly part of each day. Such individuals identified as having early-stageinflammatory disease can then be treated, advantageously, with DHCQ, orwith a synergistic combination of DHCQ and a statin, as describedherein.

In certain embodiments, this invention is directed to the treatment ofindividuals with established inflammatory disease or disease associatedwith inflammation. The inflammatory disease is diagnosed based on anindividual exhibiting symptoms, signs, clinical features, laboratorytest results, imaging test results, biomarker results, and otherfindings that enable a physician to formally diagnose that individualwith the inflammatory disease. In some embodiments, establishedinflammatory disease is an inflammatory disease for which an individualhas had a formal diagnosis of the disease made by a physician for longerthan 6 months. In established inflammatory disease, the signs orsymptoms of disease may be more severe as compared to, for example, thesymptoms for an individual diagnosed with early-stage inflammatorydisease. In established inflammatory disease, the disease process maycause tissue or organ damage. As described herein, in certainembodiments, determination of inflammation in an individual withestablished disease can comprise analyzing the individual for thepresence of at least one marker indicative of the presence ofinflammation.

Determination of inflammation in an individual at risk for, withearly-stage, or with established disease can comprise analyzing theindividual for the presence of at least one marker indicative of thepresence of inflammation or metabolic abnormalities. Markers ofinflammation include molecular markers, metabolic markers, clinicalmarkers and imaging markers. In some embodiments, the method comprisesanalyzing an individual for the presence of one, two, three, four, ormore markers of inflammation that are diagnostic for inflammation, whichcan be systemic or localized inflammation. In some embodiments at leastone of the marker(s) of inflammation is an imaging marker, includingwithout limitation radiographic imaging, ultrasound imaging, magneticresonance imaging (MRI), computed tomography (CT), etc. In someembodiments at least one of the marker(s) of inflammation is a molecularmarker, where a biological sample is obtained from the individual andanalyzed for the presence of a molecule, e.g. high-sensitivity CRP (orregular CRP), cytokine, serum amyloid A, serum amyloid P, fibrinogen,antibody (autoantibody or anti-microbial antibody), cartilage component,protease, RNA sequence (for example, mRNA encoding one or more cytokinesor other immune molecules), etc. and compared to a control or referencevalue, wherein altered level of the molecular marker is indicative ofinflammation. In some embodiments, the marker of inflammation is anabnormal clinical marker. Examples of abnormal clinical markers ofinflammation include swelling on physical examination, tenderness onphysical examination, and combinations thereof. In some embodiments themarker indicative of inflammation indicates the presence of localinflammation, i.e. inflammation present at the affected joint, in thecentral nervous system, or in another tissue, organ or site within thebody. In some embodiments, the marker of inflammation is an abnormalmetabolic marker, and the abnormal metabolic marker is selected from thegroup consisting of a blood pressure of about 140/90 mmHg or more,plasma triglyceride levels of about 1.7 mmol/L or more, high-densitylipoprotein cholesterol (HDL-C) levels of about 0.9 mmol/L or less formales and about 1.0 mmol/L or less for females, amicroalbuminuria:urinary albumin excretion ratio of about 20 μg/min ormore, or an albumin:creatinine ratio of about 30 mg/g or more, a fastingblood glucose greater than about 100 mg/dL, 2 hour post-prandial bloodglucose greater than about 200, or a hemoglobin A1c greater than about6.5 mg/dL. The measurement or detection of an abnormal inflammatory ormetabolic marker in an individual is predictive for that individualbeing at increased risk of developing, in the pre-clinical phase of, inthe early-stages of, or having an established inflammatory disease ordisease associated with inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1A-1B provides the chemical structures of hydroxychloroquine (HCQ),desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), andbisdesethylchloroquine (BDCQ).

FIG. 2 is a plot of “arthritis scores” versus “days after boost” (secondimmunization to induce arthritis) in a mouse model for rheumatoidarthritis, comparing prevention of disease with vehicle (control), HCQ,DHCQ, DCQ, and BDCQ.

FIG. 3 is a plot of “mean clinical scores” versus “time afterimmunization”, comparing vehicle, HCQ, BDCQ, and DHCQ for preventingdevelopment of mouse multiple sclerosis.

FIG. 4A-4B is a plot of “mean clinical scores” versus “time afterimmunization”, comparing vehicle, HCQ, and DHCQ treatment of establishedmouse multiple sclerosis.

FIG. 5 is a chart of cartilage degeneration scores, comparing vehicle,HCQ and DHCQ treatment of mouse osteoarthritis.

FIG. 6 is a chart of various parameters related to the progression ofmouse osteoarthritis, including “cartilage degeneration score”,“osteophyte score”, and “synovitis score”, comparing treatment withvehicle, HCQ, and DHCQ.

FIG. 7A-7D shows micrographs of liver tissue and charts of liver scores,AST levels and ALT levels, comparing vehicle, HCQ, and DHCQ forprevention of the development of non-alcoholic steatohepatitis (NASH) inmice with diet-induced obesity.

FIG. 8A-8D shows micrographs of liver tissue and charts of liver scores,AST levels and ALT levels, comparing vehicle, HCQ, and DHCQ treatment ofestablished non-alcoholic steatohepatitis (NASH) in mouse diet-inducedobesity.

FIG. 9A-9C shows graphs of glucose, triglyceride, and cholesterollevels, comparing vehicle, HCQ, DHCQ, BDCQ, and DCQ treatment of type IIdiabetes, hyperlipidemia and metabolic syndrome in mouse diet-inducedobesity.

FIG. 10 is a chart of TNFα levels, comparing treatment of humanperipheral blood mononuclear cells (PBMCs) stimulated withlipopolysaccharide (LPS) with varying amounts of HCQ or DHCQ.

FIG. 11 shows micrographs of in vitro cultured pigmented retinalepithelial cells, comparing exposure to vehicle (control, CTRL), HCQ,DHCQ, and BDCQ for 24 hours.

FIG. 12 is a graph comparing percentages of pigmented retinal cellsurvival, comparing exposure to vehicle, HCQ, DHCQ, and BDCQ.

FIG. 13 is a chart comparing HCQ and DHCQ accumulation in plasma, retinatissue, operated joint synovium, and non-operated joint synovium derivedfrom mice treated with either HCQ or DCHQ.

FIG. 14 is a chart comparing the ratio of HCQ and DHCQ levels in varioustissues derived from mice treated with either HCQ or DHCQ.

FIG. 15 is a chart comparing the ratio of HCQ and DHCQ levels in theretina tissue and plasma derived from mice treated with either HCQ orDHCQ.

FIG. 16 is a chart comparing the ratio of HCQ and DHCQ levels in theoperated joint synovium and retina tissue derived from mice treated witheither HCQ or DHCQ.

FIG. 17A-17C shows micrographs of retinal cell layers of mice treatedwith vehicle, HCQ, and DHCQ, showing ganglion cell layer nuclearshrinkage.

FIG. 18 is a chart comparing the number of cells in the ganglion celllayer for mice treated with vehicle, HCQ, and DHCQ.

FIG. 19 is a chart comparing cartilage degeneration scores for micetreated with vehicle, atorvastatin, HCQ, DHCQ, BDCQ, DCQ,atorvastatin+HCQ, atorvastatin+DHCQ, and atorvastatin+BDCQ.

FIG. 20 is a chart comparing cartilage degeneration scores, osteophytescores, and synovitis scores for subjects treated with atorvastatin,HCQ, DHCQ, BDCQ, DCQ, atorvastatin+HCQ, atorvastatin+DHCQ, andatorvastatin+BDCQ, as compared to subjects treated with vehicle(control).

FIG. 21 is a chart comparing cartilage degeneration scores, osteophytescores, and synovitis scores for mice treated with HCQ+atorvastatin ascompared to HCQ, HCQ+atorvastatin as compared to atorvastatin,DHCQ+atorvastatin as compared to DHCQ, and DCHQ+atorvastatin as comparedto atorvastatin.

FIG. 22 shows heatmap panels representing levels of inflammatorycytokines in synovial tissues derived from mice with osteoarthritistreated with vehicle (CTRL), HCQ, DHCQ, atorvastatin (Atorva),HCQ+atorvastatin, and DHCQ+atorvastatin.

FIG. 23A-23J shows charts comparing levels of inflammatory cytokines forvarious treatments.

FIG. 24A-241 shows heatmap panels comparing levels of inflammatorycytokines for various treatments.

FIG. 25A-25D shows various scores related to inflammation and painobserved for subjects with medial-compartment knee osteoarthritis in a16-week open-label clinical trial (NCT01645176).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Many diseases have an underlying inflammatory component that contributesto disease initiation and/or progression. Thus, the spectrum ofinflammatory diseases and diseases associated with inflammation is broadand includes autoimmune diseases such rheumatoid arthritis (RA),systemic lupus erythematosus (SLE), multiple sclerosis (MS), andautoimmune hepatitis; degenerative diseases such as osteoarthritis (OA),Alzheimer's disease (AD), and macular degeneration; chronic infectionssuch as human immunodeficiency virus (HIV), chronic hepatitis C virus(HCV) infection, chronic hepatitis B virus (HBV), chroniccytomegalovirus (CMV) infection, tuberculosis (TB) infection, as well asother chronic viral and bacterial infections; metabolic diseasesincluding type II diabetes, metabolic syndrome, non-alcoholicsteatohepatitis (NASH), and alcoholic steatohepatitis; cardiovasculardiseases such as atherosclerosis; cancers which can arise from andinduce inflammation; as well as other diseases with an inflammatorycomponent.

Additional inflammatory diseases and diseases associated withinflammation include but are not limited to acne vulgaris, acnecongloblata, acne fulminans, asthma, celiac disease, chronicprostatitis, ulcerative colitis, microscopic colitis, collagenouscolitis, Crohn's disease, atopic dermatitis, diverticulitis,glomerulonephritis, interstitial cystitis, viral hepatitis including butnot limited to hepatitis B and hepatitis C, interstitial cystitis,irritable bowel syndrome, reperfusion Injury, sarcoidosis, amyloidosis,and transplant rejection including but not limited to heart, lung,kidney, pancreas, bone marrow, stem cell, skin, corneal, and islet celltransplants. Additional inflammatory diseases and diseases associatedwith inflammation include cancers and pre-cancerous states and theassociated inflammatory responses. Additional inflammatory diseases anddiseases associated with inflammation include infectious diseasesassociated with inflammation which include but are not limited tochronic infection with human immunodeficiency virus (HIV), hepatitis Cvirus (HCV), hepatitis B virus (HBV), syphilis, rickettsial diseases,lyme disease, bacterial cellulitis, chronic fungal infection,ehrlichiosis, HHV-6, Herpes simplex virus 1 and 2, stongyloidiasis,Epstein barr virus, cytomegalovirus, mycoplasma infection,Creutzfeldt-Jacob disease, oncocerciasis, nocardia, Whipples disease,mycobacterial disease, tinea infection, and alphaviruses including butnot limited to chikungunya, ross river virus, or other alphaviruses.Additional inflammatory diseases and diseases associated withinflammation include but are not limited to anti-phosholipid syndrome,Hashimoto's thyroiditis, Dequervains thyroiditis, Graves thyroiditis,adrenalitis, type I diabetes mellitus, hypophysitis, pemphigus vulgaris,bullous pemphigoid, Eaton Lambert syndrome, myasthenia gravis, Addison'sdisease, ankylosing spondylitis, alopecia aureate, autoimmune hemolyticanemia, immune thrombocytopenic purpura, autoimmune hepatitis, Behcetsdisease, cardiomyopathy, chronic fatigue syndrome, chronic inflammatorydemyelinating polyneuropathy, autoimmune inner ear disease, cicatricialpemphigoid, Dego's Disease, dermatomyositis/juvenile dermatomyositis,polymyositis, inclusion body myositis, Guillain-Barre syndrome,Meniere's Disease, mixed connective tissue disease, pernicious anemiavasculitis, polychondritis, polyglandular autoimmune syndrome,polymyalgia rheumatic, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynaud's phenomenon, Reiter's syndrome, reactive arthritis,rheumatic fever, scleroderma, Sjogren's syndrome, stiff-man syndrome,Takayasu arteritis, temporal arteritis/giant cell arteritis,polyartereitis nodosa, uveitis, vitiligo, autoimmune Wilsons disease,bleeding disorders due to autoreactivity against clotting factors,chronic urticaria, vasculitis including but not limited togranulomatosus with polyangiitis, eosinophilic granulomatosis withpolyangiits, microscopic polyangiits, henoch schonlien purpura,hypersensitivity vasculitis, hypocomplementemic urticarial vasculitis,polyarteritis nodosa.

In one classification, an inflammatory disease is considered a diseasewhich exhibits clinical manifestations (abnormal clinical markers) suchas visible inflammation including pain, swelling, warmth, and redness.This classification of inflammatory disease would include, but certainlynot be limited to, RA, SLE, NAFLD, NASH, MS, metabolic syndrome, type IIdiabetes, atherosclerosis, cardiovascular disease and OA.

Diseases associated with inflammation have in their underlying pathologya focal, multifocal, or systemic inflammatory process, but which aremanifested as organ or system dysfunction without apparent clinicalinflammation. In this classification, hyperlipidemia and insulinresistance are referred to as diseases associated with inflammation.Conditions such as hyperlipidemia can be considered a disease hereinbecause it is a metabolic abnormality associated potential pathology.Likewise, insulin resistance can be considered a disease herein becauseit is a metabolic abnormality associated potential pathology.Hyperlipidemia and insulin resistance can thus be considered diseasesassociated with inflammation. It should be noted that a significantproportion of patients with hyperlipidemia, insulin resistance, with orwithout frank NASH or its precursor NAFLD, have what is known as themetabolic syndrome. The metabolic syndrome refers to a group of factors,including hypertension, obesity, hyperlipidemia, and insulin resistance(manifesting as frank diabetes or high fasting blood glucose or impairedglucose tolerance), that raises the risk of developing heart disease,diabetes, or other health problems; (Grundy et al, Circulation. 2004;109:433-438). The metabolic syndrome has been associated withinflammation both as an effect of the syndrome and as a contributor toits initiation, progression, and ultimate pathogenesis. (Romeo G R etal, Arterioscler Thromb Vasc Biol. 2012 32(8):1771-6; de Luca C et al,FEBS Lett. 2008 582(1):97-105; Ma K et al, Diabetes Metab Res Rev. 201228(5):388-94). In part, this is due to macrophage accumulation in obeseadipose tissue, where they produce TNF and other inflammatory cytokinesin response to stimulation with saturated fatty acids and circulatinglipopolysaccharide (LPS) (Johnson et al, Cell 2013. 152(4):673-84;Bhargava P et al, Biochem J. 2012 442(2):253-62). Moreover, TNFinhibition can abrogate insulin resistance (Johnson et al, Cell 2013.152(4):673-84). However, the risks of long term TNF inhibition aresignificant and require injection therapy. The identification of a safeand effective agent to ameliorate adipose macrophage activation andcytokine production would be beneficial. As many anti-inflammatory andimmunosuppressive agents are themselves associated with adversemetabolic risk (i.e. hypertension, insulin resistance, hyperlipidemia,and/or hepatic steatosis), if such an agent had a favorable effect onmetabolic profile this would be highly advantageous.

A variety of therapeutics exist for inflammatory diseases, and examplesinclude: non-steroidal anti-inflammatory drugs (NSAIDs) such asibuprofen, Naprosyn and aspirin; steroids such as prednisone andmethylprednisolone; small molecule drugs that inhibit immune cellproliferation including methotrexate, sulfasalazine, imuran,cyclophosphamide; small molecule kinase inhibitors including imatinib,tofacitinib, and others; antibody therapeutics targeting TNF(adalimumab, infliximab, etc), IL-6 (tocilizumab which targets the IL-6receptor), IL-1 (canakinumab), IL-17 (secukinumab), IL-12p40(ustekinumab), and others; non-antibody biological therapeuticsincluding CTLA4-Ig, etanercept, and IL-1 receptor antagonist(anakinura). It is well known that these drugs exhibit differentialtherapeutic activity against different inflammatory diseases. Forexample, anti-TNF antibodies effectively treat rheumatoid arthritis(RA), psoriasis and ankylosing spondylitis; but are not effective attreating systemic lupus erythematosus (SLE) and vasculitis; exacerbatemultiple sclerosis; are appropriate to use in RA and psoriasis patientswith chronic hepatitis C virus infection, but are contraindicated inpatients with chronic hepatitis B virus infection due to theirpropensity to trigger hepatitis B viral replication and exacerbation ofthe infection. In contrast, ustekinumab effectively treats psoriasis,but does not provide benefit in rheumatoid arthritis or multiplesclerosis. In contrast, methotrexate exhibits efficacy in rheumatoidarthritis and psoriasis, but does not treat SLE or Crohn's disease. As aresult, it is not possible to predict for which inflammatory diseases ordiseases associated with inflammation a particular anti-inflammatorydrug will provide a benefit. Not only does the clinical effectiveness ofanti-inflammatory drugs vary widely depending upon the indication, butalso particular anti-inflammatory drugs can exacerbate other symptoms ofthe disease. Accordingly, each anti-inflammatory drug candidate must beempirically tested and demonstrated to provide efficacy for treating aspecific inflammatory disease in order to establish the candidateanti-inflammatory drug as a safe, appropriate, and efficacious treatmentfor that particular inflammatory disease.

Rheumatoid Arthritis (RA) is a chronic syndrome characterized usually bysymmetric inflammation of the peripheral joints, potentially resultingin progressive destruction of articular and periarticular structures,with or without generalized manifestations (Firestein (2003) Nature423(6937):356-61; Mclnnes and Schett. (2011) N Engl J Med.365(23):2205-19). The cause is unknown. A genetic predisposition hasbeen identified, and, in some populations, localized to a pentapeptidein the HLA-DR beta1 locus of class II histocompatibility genes.Environmental factors may also play a role. For example, cigarettesmoking places individuals possessing HLA-DR4 containing the “sharedepitope” polymorphism at approximately 10-20 fold increased risk ofdeveloping RA. Cigarette smoking is thought to induce anti-citrullinatedprotein antibody (ACPA) responses, which are measured using thecommercial cyclic-citrullinated peptide (CCP) assay (Klareskog et al.(2006) Arthritis Rheum. 54(1):38-46). In addition, periodontitis andinfection with P. gingivalis might also play a role in the initiation ofautoimmune responses that result in development of RA (Rutger andPersson. 2012, J Oral Microbiol. 4). Immunologic changes may beinitiated by multiple factors. About 0.6% of all populations areaffected, women two to three times more often than men. Onset may be atany age, most often between 25 and 50 yr.

Prominent immunologic abnormalities that may be important inpathogenesis include antibodies and immune complexes found in jointfluid cells and in vasculitis. Plasma cells produce antibodies thatcontribute to these complexes. Lymphocytes that infiltrate the synovialtissue are primarily T helper cells, which can produce pro-inflammatorycytokines. Macrophages and their cytokines (e.g., tumor necrosis factor,granulocyte-macrophage colony-stimulating factor) are also abundant indiseased synovium. Increased adhesion molecules contribute toinflammatory cell emigration and retention in the synovial tissue.Increased macrophage-derived lining cells are prominent along with somelymphocytes and vascular changes in early disease.

In chronically affected joints, the normally delicate synovium developsmany villous folds and thickens because of increased numbers and size ofsynovial lining cells and colonization by lymphocytes and plasma cells.The lining cells produce various materials, including collagenase andstromelysin, which can contribute to cartilage destruction;interleukin-1, which stimulates lymphocyte proliferation; andprostaglandins. The infiltrating cells, initially perivenular but laterforming lymphoid follicles with germinal centers, synthesizeinterleukin-2, other cytokines, RF, and other immunoglobulins. Fibrindeposition, fibrosis, and necrosis also are present. Hyperplasticsynovial tissue (pannus) may erode cartilage, subchondral bone,articular capsule, and ligaments. PMNs are not prominent in the synoviumbut often predominate in the synovial fluid.

Onset is usually insidious, with progressive joint involvement, but maybe abrupt, with simultaneous inflammation in multiple joints. Tendernessin nearly all inflamed joints is the most sensitive physical finding.Synovial thickening, the most specific physical finding, eventuallyoccurs in most involved joints. Symmetric involvement of small handjoints (especially proximal interphalangeal and metacarpophalangeal),foot joints (metatarsophalangeal), wrists, elbows, and ankles istypical, but initial manifestations may occur in any joint. RA ischaracterized by the development of focal bone erosions throughdegradation and remodeling of bone at the joint margins and insubchondral bone of patients with RA. A hallmark of a subset of RA isthe development of autoantibodies, including rheumatoid factors (RF) andanti-citrullinated protein antibodies (ACPA). RF, antibodies to humanγ-globulin, are present in about 70% of patients with RA. However, RF,often in low titers, occurs in patients with other diseases, includingother connective tissue diseases such as systemic lupus erythematous,granulomatous diseases, chronic infections such as viral hepatitis,subacute bacterial endocarditis, and tuberculosis, and cancers. Low RFtiters can also occur in a small percentage of the general population,and more commonly in the elderly. Another disease indicator is thepresence of ACPA, which are measured using the clinical anti-CCP (cycliccitrullinated peptide) antibody test. Anti-CCP antibodies areapproximately 60% sensitive and 95% specific for the diagnosis of RA,and like RF, predict a worse prognosis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune diseasecharacterized by malar rashes, oral ulcers, photosensitivity, serositis,seizures, low white blood cell counts, low platelet counts, seizures, apositive anti-nuclear antibody (ANA) test, and other positiveautoantibodies. SLE is an autoimmune disease characterized by polyclonalB cell activation, which results in a variety of anti-protein andnon-protein autoantibodies that result in immune complexes andinflammation which contributes to tissue damage (see, e.g., Kotzin etal., 1996, Cell 85:303-06 for a review of the disease). SLE has avariable course characterized by exacerbations and remissions and isdifficult to study. For example, some patients may demonstratepredominantly skin rash and joint pain, show spontaneous remissions, andrequire little medication. The other end of the spectrum includespatients who demonstrate severe and progressive kidney involvement(glomerulonephritis and cerebritis) that requires therapy with highdoses of steroids and cytotoxic drugs such as cyclophosphamide.Hydroxychloroquine slows SLE progression, and is a mainstay therapeuticfor the management of SLE.

Multiple sclerosis (MS) is a debilitating, inflammatory, neurologicalillness characterized by demyelination of the central nervous system.The disease primarily affects young adults with a higher incidence infemales. Symptoms of the disease include fatigue, numbness, tremor,tingling, dysesthesias, visual disturbances, dizziness, cognitiveimpairment, urological dysfunction, decreased mobility, and depression.Four types classify the clinical patterns of the disease:relapsing-remitting, secondary progressive, primary-progressive andprogressive-relapsing (S. L. Hauser and D. E. Goodkin, MultipleSclerosis and Other Demyelinating Diseases in Harrison's Principles ofInternal Medicine 14th Edition, vol. 2, Mc Graw-Hill, 1998, pp.2409-19).

Inflammatory bowel diseases, include Crohn's disease and ulcerativecolitis, involve autoimmune attack of the bowel. These diseases causechronic diarrhea, frequently bloody, as well as symptoms of colonicdysfunction.

Systemic sclerosis (SSc, or scleroderma) is an autoimmune diseasecharacterized by fibrosis of the skin and internal organs and widespreadvasculopathy. Patients with SSc are classified according to the extentof cutaneous sclerosis: patients with limited SSc have skin thickeningof the face, neck, and distal extremities, while those with diffuse SSchave involvement of the trunk, abdomen, and proximal extremities aswell. Internal organ involvement tends to occur earlier in the course ofdisease in patients with diffuse compared with limited disease (Laing etal. (1997) Arthritis. Rheum. 40:734-42). The majority of patients withdiffuse SSc who develop severe internal organ involvement will do sowithin the first three years after diagnosis at the same time the skinbecomes progressively fibrotic (Steen and Medsger (2000) ArthritisRheum. 43:2437-44.). Common manifestations of diffuse SSc that areresponsible for substantial morbidity and mortality include interstitiallung disease (ILD), Raynaud's phenomenon and digital ulcerations,pulmonary arterial hypertension (PAH) (Trad et al. (2006) Arthritis.Rheum. 54:184-91), musculoskeletal symptoms, and heart and kidneyinvolvement (Ostojic and Damjanov (2006) Clin. Rheumatol. 25:453-7).Current therapies focus on treating specific symptoms, butdisease-modifying agents targeting the underlying pathogenesis arelacking.

Autoimmune hepatitis is a disease in which the body's immune systemattacks liver cells. This immune response causes inflammation of theliver, also called hepatitis. Researchers think a genetic factor maymake some people more susceptible to autoimmune diseases. About 70percent of those with autoimmune hepatitis are female. The disease isusually quite serious and, if not treated, gets worse over time.Autoimmune hepatitis is typically chronic, meaning it can last foryears, and can lead to cirrhosis—scarring and hardening—of the liver.Eventually, liver failure can result.

Four subtypes of autoimmune hepatitis are recognized, but the clinicalutility of distinguishing subtypes is limited. (1) positive ANA and SMA,elevated immunoglobulin G (classic form, responds well to low dosesteroids); (2) positive LKM-1 (typically female children and teenagers;disease can be severe), LKM-2 or LKM-3; (3) positive antibodies againstsoluble liver antigen (this group behaves like group 1) (anti-SLA,anti-LP), and (4) no autoantibodies detected (˜20%) (of debatablevalidity/importance) (Krawitt et al. Autoimmune hepatitis. New EnglandJournal of Medicine, 1996 334 (14): 897-903).

Many degenerative diseases have an underlying inflammatory component,and examples of such degenerative diseases include osteoarthritis (OA),Alzheimer's disease (AD), and macular degeneration.

Osteoarthritis (OA) affects nearly 27 million people in the UnitedStates, accounting for 25% of visits to primary care physicians, andhalf of all NSAID prescriptions. Rheumatoid arthritis (RA) is anautoimmune synovitis that affects approximately 0.6% of the worldpopulation. OA is a chronic arthropathy characterized by disruption andpotential loss of joint cartilage along with other joint changes,including bone remodeling that may include bone hypertrophy (osteophyteformation), subchondral sclerosis, and formation of subchondral cysts.OA is viewed as failure of the synovial joint (Abramson et al, ArthritisRes Ther. 2009; 11(3):227; Krasnokutsky et al, Osteoarthritis Cartilage.2008; 16 Suppl 3:S1-3; Brandt et al, Rheum Dis Clin North Am. 2008August; 34(3):531-59). OA results in the degradation of joints,including articular cartilage and subchondral bone, resulting inmechanical abnormalities and impaired joint function. Symptoms mayinclude joint pain, tenderness, stiffness, sometimes an effusion, andimpaired joint function. A variety of causes can initiate processesleading to loss of cartilage.

OA may begin with joint damage from trauma to the joint; mechanicalinjury to the meniscus, articular cartilage, a joint ligament, oranother joint structure; defects in cartilage matrix components; and thelike. Mechanical stress on joints may underlie the development of OA inmany individuals, with many and varied sources of mechanical stress,including misalignments of bones caused by congenital or pathogeniccauses; mechanical injury; overweight; loss of strength in musclessupporting joints; and impairment of peripheral nerves, leading tosudden or dyscoordinated movements that overstress joints.

Articular cartilage comprises chondrocytes that generate and aresurrounded by extracellular matrix. In synovial joints there are atleast two movable bony surfaces that surrounded by the synovialmembrane, which secretes synovial fluid, a transparent alkaline viscidfluid which fills the joint cavity, and articular cartilage, which isinterposed between the articulating bony surfaces. The earliest grosspathologic finding in osteoarthritis is softening of the articularcartilage in habitually loaded areas of the joint surface. Thissoftening or swelling of the articular cartilage is frequentlyaccompanied by loss of proteoglycans from the cartilage matrix. Withprogression of osteoarthritis the integrity of the cartilage surface islost and the articular cartilage thins, with vertical clefts extendinginto the depth of the cartilage in a process called fibrillation. Jointmotion may cause fibrillated cartilage to shed segments that expose thebone underneath (subchondral bone). The subchondral bone is remodeled inOA, including the development of subchondral sclerosis, development ofsubchondral cysts, and the formation of ectopic bone termed osteophytes.Subchondral cysts also develop which may be filled with synovial fluid.At the joint margins osteophytes (bone spurs) form. The remodeling ofsubchondral bone increases the mechanical strain and stresses on boththe overlying articular cartilage and subchondral bone, leading tofurther damage of both the cartilage and subchondral bone.

The tissue damage stimulates chondrocytes to attempt repair, whichincreases production of proteoglycans and collagen. However, efforts atrepair also stimulate the enzymes that degrade cartilage, as well asinflammatory cytokines, which are normally present in small amounts.Inflammatory mediators trigger an inflammatory cycle that furtherstimulates the chondrocytes and synovial lining cells, eventuallybreaking down the cartilage. Chondrocytes undergo programmed cell death(apoptosis).

OA is characterized pathologically by low-grade infiltration ofinflammatory cells including inflammatory cells, primarily macrophages,but also B cells and T cells. These cells, again primarily macrophages,are capable of producing inflammatory cytokines and MMPs in the OAjoint. However, when stimulated by inflammatory cytokines including IL-1and TNF, primary cells within the joint, including synovial fibroblastsand chondrocytes, are capable of producing further cytokines includingIL-6 as well as multiple MMPs.

OA should be suspected in patients with gradual onset of symptoms andsigns, particularly in older adults, usually beginning with one or a fewjoints. Pain can be the earliest symptom, sometimes described as a deepache. Pain is usually worsened by weight bearing and relieved by restbut can eventually become constant. Stiffness follows awakening orinactivity. If OA is suspected, plain x-rays should be taken of the mostsymptomatic joints. X-rays generally reveal marginal osteophytes,narrowing of the joint space, increased density of the subchondral bone,subchondral cyst formation, bony remodeling, and joint effusions (whichare considered abnormal imaging markers). Standing x-rays of knees aremore sensitive in detecting joint space narrowing. Magnetic resonanceimaging (MRI) can be used to detect cartilage degeneration, and severalMRI-based based scoring systems have been developed to characterize theseverity of OA (Hunter et al, PM R. 2012 May; 4(5 Suppl):S68-74).

OA commonly affects the hands, feet, spine, and the large weight bearingjoints, such as the hips and knees, although in theory, any joint in thebody can be affected. As OA progresses, the affected joints appearlarger, are stiff and painful, and usually feel better with gentle usebut worse with excessive or prolonged use. Treatment generally involvesa combination of exercise, lifestyle modification, and analgesics. Ifpain becomes debilitating, joint replacement surgery may be used toimprove the quality of life.

Among the agents proposed to provide for disease modification, such asdoxycycline (presumably though its action at an MMP inhibitor),bisphosphonates (presumably aimed at inhibiting osteoclast activation)and licofelone (by inhibiting the cyclooxygenase and lipoxegenasepathways), none have demonstrated robust chondroprotection as defined byslowing of cartilage breakdown. Among the agents that have demonstratedpartial efficacy in control of pain associated with OA are analgesicssuch as acetaminophen and anti-inflammatories including non-steroidalanti-inflammatory agents (NSAIDs), opiates, intraarticularcorticosteroids, and hyaluronic acid derivatives injected into thejoint. These agents have not been demonstrated to prevent cartilage lossor slow the loss of joint function.

Given the slow progression seen in OA, the need to take an agent forlengthy periods of time necessitates a high degree of safety. Thus,there is need for therapeutic options that provide disease modifyingfunctional activity and a safety profile that allows an extendedduration of therapy.

Murine models of OA include induction of OA through destabilization ofthe medial meniscus (DMM) or medial meniscectomy (MM). Histologicalanalysis of stifle (knee) joint articular cartilage at serial timepoints after medial meniscectomy in the MM model. Approximately 2-6months following surgical induction, mice are sacrificed and histologicsections stained with toluidine-blue, Safranin-O, and/or hematoxylin andeosin (H&E) to determine the level of cartilage loss (or level ofcartilage degeneration, or “OA score”), as well as the degree ofosteophyte formation, and the degree of synovial inflammation (termedsynovitis).

Alzheimer's disease (AD) is the most common neurodegenerative disease inthe population (Cummings et al., Neurology 51, S2-17; discussion S65-7,1998). AD affects approximately 10% of people over age 65 and almost 50%of people over age 85. It is estimated that by the year 2025, about 22million individuals will be afflicted with AD. AD is characterized by aslowly progressive dementia. The definitive diagnosis of AD is made ifthe triad of dementia, neurofibrillary tangles, and senile plaques arefound post-mortem. Senile plaques are invariably found in the brains ofpatients with Alzheimer disease. The principal constituent of senileplaques is amyloid beta protein (A3) (Iwatsubo et al., Neuron 13:45-53,1994) (Lippa et al., Lancet 352:1117-1118, 1998). AP is a 42 amino acidpeptide that is derived from the amyloid precursor protein (APP), whichis a transmembrane glycoprotein with a variety of physiologic roles,including cell proliferation, adhesion, cell signaling, and neuriteoutgrowth (Sinha et al., Ann N Y Acad Sci 920:206-8, 2000). APP isnormally cleaved within the Aβ domain to generate a secreted fragment.However, alternative processing leads to the cleavage of APP to generatesoluble Aβ that can accumulate within senile plaques. Currentlyavailable drugs are central cholinesterase inhibitors aimed atincreasing the concentration of postsynaptic acetylcholine in the brain(Farlow and Evans, Neurology 51, S36-44; discussion S65-7, 1998); (Hake,Cleve Clin J Med 68, 608-9:613-4, 616, 2001). These drugs provideminimal clinical benefit in only a few cognitive parameters.

Macular degeneration can be of the wet type related to retinalneovascularization and vascular leak but is more commonly of the drytype also known as age-related macular degeneration (AMD). AMD is achronic disease associated with loss of central vision, blurred vision,and ultimately blindness. Though the causes and risk factors for AMD aremultifactorial, activation of innate immunity involving complementactivation as well as cytokine production by macrophage and microgliahas been implicated in development of AMD. Anti-inflammatory therapyincluding corticosteroids, non-steroidal anti-inflammatory agents,methotrexate, rapamycin, and biologic agents including TNF inhibitorsand complement inhibitors have been suggested to slow progression of AMD(Wang et al, 2011. Eye (2011)25, 127-139). However, because thesetreatments are not curative and AMD is a chronic, non-fatal disease,their use is limited by risk of toxicity.

The HIV virus is the cause of AIDS, a nearly uniformly fatal diseasewithout treatment. However, the advent of highly active antiretroviraltherapy (HAART) has resulted in the change of HIV from fatal disease toa chronic condition. With the prolonged lifespan of individuals withHIV, it has been noted that despite viral suppression and even immunereconstitution as measured by peripheral CD4 T cell counts, there isstill an increased morbidity and mortality which is primary due tometabolic derangements and increased cardiovascular risk. The exactsource of this immune activation not been determined, but the continuedlow grade replication of HIV virus and activation of the endosomal TLR7receptor as well as activation of CD8 T cell response have beenimplicated. Additionally, irreversible damage to the immune cells of gutmucosa result in increased bacterial and endotoxin translocation andthus systemic inflammation (Deeks 2011 Annu Rev Med. 62:141-55). Asexpected, levels of cytokines including TNF, IL-6 as well as otherinflammatory markers such as CRP as well as coagulation markers such asD-dimer are notably elevated despite successful HAART therapy (Deeks2011. Annu Rev Med. 62:141-55).

Other chronic infections can also cause persistent inflammation. Suchinfections include chronic hepatitis B virus infection, chronichepatitis C virus infection, cytomegalovirus (CMV) infection, herpessimplex virus (HSV) infection, Epstein Barr virus (EBV) infection,chronic pseudomonas infection, chronic Staphylococcus infection, andother chronic viral, bacterial, fungal, parasitic, and other infections.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholicsteatohepatitis (NASH) are conditions associated with fatty infiltrationof the liver. NAFLD is one cause of a fatty liver, occurring when fat isdeposited (steatosis) in the liver not due to excessive alcohol use(Clark J M et al, J. American Medical Association 289 (22): 3000-4,2003). It can be related to insulin resistance and the metabolicsyndrome and may respond to treatments originally developed for otherinsulin-resistant states (e.g. diabetes mellitus type 2) such as weightloss, metformin and thiazolidinediones.

NAFLD is considered to cover a spectrum of disease activity. Thisspectrum begins as fatty accumulation in the liver (hepatic steatosis).A liver can remain fatty without disturbing liver function, but byvarying mechanisms and possible insults to the liver may also progressto become NASH, a state in which steatosis is combined with inflammationand fibrosis. NASH is a progressive disease: over a 10-year period, upto 20% of patients with NASH will develop cirrhosis of the liver, and10% will suffer death related to liver disease. NASH is the most extremeform of NAFLD, and is regarded as a major cause of cirrhosis of theliver of unknown cause (McCulough A J et al, Clinics in Liver Disease 8(3): 521-33, 2004).

Common findings in NAFLD and NASH are elevated liver enzymes and a liverultrasound showing steatosis. An ultrasound may also be used to excludegallstone problems (cholelithiasis). A liver biopsy (tissue examination)is the only test widely accepted as definitively distinguishing NASHfrom other forms of liver disease and can be used to assess the severityof the inflammation and resultant fibrosis (Adams L A et al, PostgradMed J 82(967):315-22, 2006). Non-invasive diagnostic tests have beendeveloped, such as FibroTest, that estimates liver fibrosis, andSteatoTest, that estimates steatosis, however their use has not beenwidely adopted (McCulough A J et al, Clinics in Liver Disease 8 (3):521-33, 2004).

Although fatty infiltration alone does not cause liver damage, when itis accompanied by an inflammatory reaction it can lead to fibrosis andliver cirrhosis and ultimately hepatic failure. The inflammation in NASHis characterized by infiltration of the liver by macrophages andlymphocytes, as well as alterations in the liver's macrophage-likeKupfer cell population (Tilg, et al, 2010. Hepatology. 52(5):1836-46).Inflammatory cytokines, particularly TNF, are central to the pathologyof NASH. The source of TNF is unclear: it may be peripheral, i.e.,inflammatory adipose tissue, or local, i.e., innate immune cellsactivated by portal-derived endotoxin or by free fatty acid. Theendotoxin-responsive TLR4 receptor has been shown to be critical todisease in a mouse model of NASH (Tsukumo et al, Diabetes 2007.56(8):1986-98).

A large number of treatments for NAFLD and NASH have been studied.Treatment approaches include: (i) Treatment of nutrition and excessivebody weight, (ii) weight loss, (iii) weight loss surgery, (iv) insulinsensitizers including metformin and thiazolidinediones, (v) Vitamin Ecan improve some symptoms, (vi) statins have been shown to improve liverbiochemistry and histology in patients with NAFLD; McCulough A J et al,supra; Chalasani N. et al, Gastroenterology 142(7):1592-1609, 2012).

Type II diabetes mellitus and metabolic syndrome. Type II diabetesmellitus is characterized by insulin resistance and hyperglycemia, whichin turn can cause retinopathy, nephropathy, neuropathy, or othermorbidities. Additionally, diabetes is a well-known risk factor foratherosclerotic cardiovascular disease. Metabolic syndrome refers to agroup of factors, including hypertension, obesity, hyperlipidemia, andinsulin resistance (manifesting as frank diabetes or high fasting bloodglucose or impaired glucose tolerance), that raises the risk ofdeveloping heart disease, diabetes, or other health problems; (Grundy etal, Circulation. 2004; 109:433-438). There is a well-characterizedprogression from normal metabolic status to a state of impaired fastingglucose (IFG: fasting glucose levels greater than 100 mg/dL) or to astate of impaired glucose tolerance (IGT: two-hour glucose levels of 140to 199 mg/dL after a 75 gram oral glucose challenge). Both IFG and IGTare considered pre-diabetic states, with over 50% of subjects with IFGprogressing to frank type II diabetes within, on average, three years(Nichols, Diabetes Care 2007. (2): 228-233). The insulin resistance iscaused, at least in part, by chronic low-grade inflammation (Romeo G Ret al, Arterioscler Thromb Vasc Biol. 2012 32(8):1771-6; de Luca C etal, FEBS Lett. 2008 582(1):97-105; Ma K et al, Diabetes Metab Res Rev.2012 28(5):388-94). Macrophages accumulate in obese adipose tissue,where they produce TNF and other inflammatory cytokines in response tostimulation with saturated fatty acids and circulatinglipopolysaccharide (LPS) (Johnson et al, Cell 2013. 152(4):673-84;Bhargava P et al, Biochem J. 2012 442(2):253-62). Moreover, TNFinhibition can abrogate insulin resistance (Johnson et al, Cell 2013.152(4):673-84).

Hyperlipidemia is considered a disease herein because it is a metabolicabnormality associated potential pathology. Hyperlipidemia involvesabnormally elevated levels of any or all lipids and/or lipoproteins inthe blood. The term lipids includes cholesterol and triglycerides. Thereare many different types of lipid (also called lipoproteins). Bloodtests can measure the levels of lipoproteins. The standard lipid bloodtests include a measurement of total cholesterol, LDL (low densitylipoproteins) and HDL (high density lipoproteins), and triglycerides.

Total cholesterol—A high total cholesterol level can increase anindividual's risk of cardiovascular disease. However, decisions aboutwhen to treat high cholesterol are usually based upon the level of LDLor HDL cholesterol, rather than the level of total cholesterol. A totalcholesterol level of less than 200 mg/dL (5.17 mmol/L) is normal. Atotal cholesterol level of 200 to 239 mg/dL (5.17 to 6.18 mmol/L) isborderline high. A total cholesterol level greater than or equal to 240mg/dL (6.21 mmol/L) is high. The total cholesterol level can be measuredany time of day. It is not necessary to fast (avoid eating for 12 hours)before testing.

LDL cholesterol—The low density lipoprotein (LDL) cholesterol (sometimescalled “bad cholesterol”) is a more accurate predictor of cardiovasculardisease than total cholesterol. Higher LDL cholesterol levels increaseyour risk of cardiovascular disease.

Most healthcare providers prefer to measure LDL cholesterol after youhave not eaten (fasted) for 12 to 14 hours. A test to measure LDL inpeople who have not fasted is also available, although the results maydiffer slightly. Increased LDL cholesterol is associated with increasedrisk of heart attack and stroke. In general, LDL levels fall into thesecategories: Less than 100 mg/dL Optimal; 100 to 129 mg/dL Near or aboveoptimal; 130 to 159 mg/dL Borderline high; 160 to 189 mg/dL High; 190mg/dL and above Very high.

The Framingham Risk Score is a gender-specific algorithm used toestimate the 10-year cardiovascular risk, including the risk ofcardiovascular events, myocardial infarction, stroke, heart failure, andother events, of an individual—such cardiovascular diseases represent aninflammatory disease and/or diseases associated with inflammation. TheFramingham Risk Score was first developed based on data obtained fromthe Framingham Heart Study, to estimate the 10-year risk of developingcoronary heart disease (Wilson et al, Prediction of coronary heartdisease using risk factor categories. 1998 Circulation 97 (18):1837-1847; D'Agostino et al, General cardiovascular risk profile for usein primary care: the Framingham Heart Study 2008. Circulation 117 (6):743-753). The Framingham risk score is based on gender andgender-specific formulas that incorporate age, total cholesterol level,cigarette smoking history, HCL cholesterol level, and systolic bloodpressure.

Atherosclerosis and atherosclerotic cardiovascular disease are diseasesof the arterial wall. They are characterized by accumulation of fattymaterials in the arterial wall, resulting in development of fattyplaques, which may rupture and cause vascular occlusion and ischemia. Ifsuch vascular occlusion and ischemia occur in a coronary artery,myocardial infarction may result. The atherosclerotic lesion comprises ahighly inflammatory milieu characterized by the accumulation ofinflammatory cells, including macrophages and to a lesser extent T and Bcells, and the production of high levels of inflammatory cytokines,chemokines, and MMPs (Libby et al, Nature 2011. 473(7347):3170-25).Atherosclerosis may also be associated with low-grade systemicinflammation, as evidenced by high levels of high-sensitivity CRP(hsCRP) in the blood, an abnormality that can be partially countered bytreatment with the drug rosuvastatin (Libby et al, Nature 2011.473(7347):3170-25).

Hydroxychloroquine (HCQ) can be a potent anti-inflammatory agent usedfor treating inflammatory diseases. However, the use of HCQ in suchtherapies is limited due to the significant risk of retinal toxicity.Thus vigilant ophthalmologic monitoring coupled with dose reduction oreven avoidance of HCQ is recommended, particularly for those atincreased risk for retinal toxicity. Individuals at increased risk forretinal toxicity include those who have taken HCQ for longer than fiveyears; have other risk factors, such as a high body fat level,concomitant kidney or liver disease or concomitant retinal disease; areolder than 60 years of age; or are particularly diminutive in sizeand/or body mass. The population of patients with concomitant retinaldisease, or who are at significant risk for development of concomitantretinal disease includes the rapidly growing population of patients withtype II diabetes and the associated metabolic syndrome. As theprevalence of diabetes is currently over 10% in most parts of the UnitedStates, and is approaching similar levels in other developed countries,this places a significant proportion of the population at increased riskof HCQ-mediated retinal toxicity. Notably, rates of type II diabetes arefurther increased among patients with inflammatory diseases for whichHCQ is currently indicated, including rheumatoid arthritis and systemiclupus (Dubreil H, Rheumatology (2014) 53 (2): 346-352.) Anotherinflammatory condition not previously treated (nor suggested to betreated) with HCQ is non-alcoholic steatohepatitis (NASH). This is aninflammatory condition associated with fatty infiltration of the liverand most commonly associated with concomitant presence of type IIdiabetes and the associated metabolic syndrome. In fact, it is estimatedthat 86% of patient with type II diabetes have NASH with nearly the sameproportion of NASH patients having type II diabetes (Verderese J P,Expert Rev Gastroenterol Hepatol. 2013 July; 7(5):405-7.) Thus, themajority of patients with NASH have type II diabetes and are atincreased risk for retinal toxicity in general, which is acontraindication for HCQ therapy. Accordingly, there is a need forimproved therapies for such patients, with decreased retinal toxicity.

The metabolites of hydroxychloroquine (HCQ) includedesethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), andbisdesethylchloroquine (BDCQ); their chemical structures are presentedin FIG. 1. Desethylhydroxychloroquine (DHCQ) is assigned CAS 4298-15-1;and also referred to as cletoquine; DESETHYL HYDROXY CHLOROQUINE.

HCQ increases lysosomal pH in antigen-presenting cells, and this isbelieved to be a primary mechanism by which it exerts anti-inflammatoryeffects and alters toll-like receptor (TLR) activity (Waller et al.Medical pharmacology and therapeutics (2nd ed.). p. 370). HCQ inhibitsTLRs on plasmacytoid dendritic cells, macrophage and other cells.Activation of TLR 9, a TLR that recognizes DNA-containing immunecomplexes, leads to the production of interferon and causes thedendritic cells to mature and present antigen to T cells. HCQ, bydecreasing TLR 9 signaling, reduces the activation of dendritic cellsand hence the inflammatory process.

As described herein, desethylhydroxychloroquine (DHCQ) exhibits similar,and in various cases superior (FIGS. 2-9, 19-21) activity in treating orpreventing inflammatory diseases compared to HCQ treatment. Further, itwas unexpected and surprising that only the DHCQ metabolite of HCQexhibited potent efficacy in preventing and treating inflammatorydiseases and diseases associated with inflammation, while the BDCQ andDCQ metabolites of HCQ did not provide activity in treating inflammatorydiseases and diseases associate with inflammation (FIGS. 1, 2, 3, 9, 19and 20). In addition to unexpectedly exhibiting efficacy in treatinginflammatory disease, DHCQ also unexpectedly and surprisingly exhibitslevels of efficacy superior to that of HCQ in treating metabolicabnormalities, specifically in reducing hyperglycemia, reducing serumlipids, and treating metabolism-induced inflammatory disease.

Despite both DHCQ and HCQ both exhibiting activity in preventing andtreating inflammatory diseases and diseases associated withinflammation, DHCQ exhibits significantly reduced retinal accumulationas well as significantly reduced toxicity to retinal cells as comparedto the retinal accumulation and toxicity observed for HCQ. The reducedretinal cell toxicity in DHCQ is unexpected and surprising since onlytreatment with the DHCQ metabolite of HCQ, and not treatment with theBDCQ metabolite of HCQ, provided reduced retinal toxicity (FIGS. 11-12).Further, the reduced retinal cell toxicity by DHCQ is unexpected andsurprising since only the DHCQ metabolite of HCQ, and not the BDCQmetabolite of HCQ, provided reduced retinal cell toxicity (FIGS. 11-12).In addition, only the DHCQ, and not the BDCQ and DCQ metabolites of HCQ,exhibited anti-inflammatory efficacy (FIGS. 2, 3, 9, 19 and 20).

Compositions and methods are provided for preventing or treatinginflammatory diseases or diseases associated with inflammation,including autoimmune diseases, degenerative diseases, metabolicdiseases, and other inflammatory diseases, by administration to anindividual in need thereof an effective dose of the aminoquinolinedesethylhydroxychloroquine (DHCQ). In further embodiments, thecompositions and methods provided herein are suitable for treatinglow-grade inflammation or ameliorating inflammation or treating diseasesassociated with inflammation or treating metabolic abnormalitiesassociated with diseases associated with inflammation. A benefit of themethods of the present invention is the ability to deliver a dose of anagent that is effective in treating inflammation while sparing theindividual from retinal accumulation and toxicity found withhydroxychloroquine (HCQ) treatment. Reduced monitoring of retinaltoxicity during treatment can be a feature of treatment with DHCQcompared to HCQ treatment. Treatment with DHCQ can be initiated earlierin the disease process, and can be maintained for more extended periodsof time compared to conventional treatment methods and agents.

In some embodiments the present invention provides compositions of DHCQor pharmaceutically acceptable salts or esters thereof; or a combinationDHCQ or pharmaceutically acceptable salts or esters thereof and one ormore second agent from a different drug class, for example a drug thatexerts distinct but overlapping mediatory effects, which compositionsare utilized to treat an inflammatory disease.

Recent clinical observations in humans demonstrated that takingconventional doses of HCQ (with 400 mg/day being a common dose), theincidence of retinal toxicity increases markedly with the duration oftherapy and occurs in approximately 1% of HCQ treated humans after 5years of treatment, and approximately 2% of HCQ treated humans after10-15 years of treatment (Marmor et al. Arthritis Care Res. 2010;62(6):775-84; Levy et al, Incidence of hydroxychloroquine retinopathy in1,207 patients in a large multicenter outpatient practice. ArthritisRheumatism 1997, 40(8):1482-6; Mavrikakis et al, The incidence ofirreversible retinal toxicity in patients treated withhydroxychloroquine: a reappraisal. Opthalmology, 2003, 110(7):1321-6).Notably, despite the observed rates of retinal toxicity, total rates ofphysician discontinuation of HCQ for earlier eye problems (includingasymptomatic changes noted on ophthalmologic examination) approach 7% oftreated patients over 5 years (Marmor et al. Rates and predictors ofhydroxychloroquine retinal toxicity in patients with rheumatoidarthritis and systemic lupus erythematosus, Arthritis Care Res. 2010;62(6):775-84).

The present data demonstrates that DHCQ surprisingly exhibitssignificantly decreased retinal accumulation as well as significantlydecreased retinal toxicity both in vivo and in vitro (see FIGS. 11-18),at dosage levels which provide clinical efficacy similar to that ofconventional treatments with HCQ. The estimated rate of retinal toxicityfor long-term treatment with DHCQ is less than 50% of the currentlyreported rate for HCQ when used at a similar effective cumulative doseand over a similar time period. More specifically, as compared to therates of retinal toxicity described by Marmor et al. for treatment withHCQ (Ophthalmology. 2011 February; 118(2):415-22), given a similar levelof therapeutic activity and time period of dosing for both HCQ and DHCQ,DHCQ is estimated to provide less than 50% of retinal toxicity ascompared to that with HCQ dosing. Thus, in patient populations analogousto that described by the American College of Opthamolology and Marmor etal (Ophthalmology. 2011 February; 118(2):415-22) where the retinaltoxicity rate approach 1% after 5 years, DHCQ therapy is estimated toreduce the retinal toxicity to less that 0.5% of treated individuals.Further, given conventional assumptions that retinal screening isjustified as rates of toxicity approach 1%, the reduced cumulative ratesof retinal toxicity associated with DHCQ therapy will substantiallyreduce the need for retinal toxicity screening to a single screening at5 and 10 years, or entirely negate the need for screening.

In the description that follows, a number of terms conventionally usedin the field of treating inflammation are utilized extensively. In orderto provide a clear and consistent understanding of the specification andclaims, and the scope to be given to such terms, the followingdefinitions are provided.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims.

As used herein the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the culture” includes reference to one or more culturesand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety forall purposes to the same extent as if each individual publication,patent, or patent application was specifically and individuallyindicated to be incorporated by reference.

Inflammatory diseases are diseases that involve inflammation. Thepresence of inflammation can be detected by a variety of approaches,including clinical history, physical examination, laboratory testing,histologic analysis of tissue, analysis of biomarkers, and imaging.Clinical features and physical exam markers of inflammation includeswelling, effusions, edema, redness, warmth, pain, or associatedpathologically with the influx of inflammatory cells or production ofinflammatory mediators. Laboratory testing and/or histologic markers areabnormal when increased numbers of inflammatory cells are demonstrated.Markers of inflammation can include a molecular marker(s), and examplesof a molecular marker(s) include C-reactive protein, a cytokine, anantibody, a DNA sequence, an RNA sequence, a cartilage marker, ametabolic marker, a bone marker, or combinations thereof. Imaging canreveal findings including enhancement of tissues, edema and swelling oftissues, and other findings indicative of inflammation. Examples ofimaging markers of inflammation can include imaging markers measuredusing magnetic resonance imaging, ultrasound, computed tomography,angiography, and combinations thereof.

The presence of low-grade inflammation is characterized by anelevation(s) in the local or systemic concentrations of cytokines suchas TNF-α, IL-6, and c-reactive protein (CRP), and occurs in adiposity,osteoarthritis, Alzheimer's disease, type II diabetes, metabolicsyndrome, coronary artery disease, nonalcoholic fatty liver disease(NAFLD), non-alcoholic steatohepatitis, and many chronic anddegenerative diseases. Low-grade inflammation is manifest byinflammation present at a level below the “high-grade” inflammationdetected in active autoimmune diseases (such as active rheumatoidarthritis, psoriasis, Crohn's disease, systemic lupus erythematous,autoimmune hepatitis, and other autoimmune states) and in certain viraland bacterial infections during which humans experience clinicalsymptoms (such as influenza virus infection, Staphylococcus aureusinfection, and other infections).

The reduction or amelioration of inflammation is indicated bydissipation of inflammation, a reduction in number of inflammatory cellsor in levels of inflammatory mediators as evidenced by symptomaticrelief (including but not limited to pain relief), radiographic changes,biochemical changes, pathologic/histologic changes, decreasedprogression of such markers of inflammation, decreased development offindings indicative of tissue or organ damage, decreased development ofsymptoms or signs of disease, or decreased development of disease.

A symptom is a departure from normal function or feeling which isnoticed by an individual, indicating the presence of disease orabnormality. A symptom is subjective, observed by the individualpatient, and cannot be measured directly.

A sign of disease or a medical sign (also termed marker(s) herein) is anobjective indication of some medical fact or characteristic that may bedetected during a physical examination, by an in vivo examination of apatient, by a laboratory test, by a radiographic or other imaging test,or by another. Signs or markers may have no meaning to the patient, andmay even go unnoticed, but may be meaningful and significant to thehealthcare provider in assisting the diagnosis of medical condition(s)responsible for the patient's symptoms. Examples of abnormal markers orsigns include elevated blood pressure (greater than about 140 mmHgsystolic and/or 90 mmHg diastolic), cholesterol (LDL greater than about140 mg/dL, triglycerides greater than about 200 mg/dL, or HDL less thanabout 40 mg/dL), a clubbing of the fingers (which may be a sign of lungdisease, or many other things), arcus senilis, loss of proteoglycans inthe cartilage, increased blood glucose, increased levels of certainmarkers of liver inflammation, elevated levels of acute phase proteinsinclude ESR greater than about 20, hsCRP greater than about 0.75 mg/L,and other findings. Signs are any indication of a medical condition thatcan be objectively observed (i.e., by someone other than the patient),whereas a symptom is merely any manifestation of a condition that isapparent to the patient (i.e., something consciously affecting thepatient). From this definition, it can be said that an asymptomaticpatient is uninhibited by a disease. However, a doctor may discover thesigns of hypertension in an asymptomatic patient who does not experience“disease”, and the sign indicates a pre-clinical or early-stage diseasestate that poses a hazard to the patient.

Administration of a drug or other chemical entity to an animal, human orother mammal includes administration via any route including but notlimited to oral, intradermal, intramuscular, intraperitoneal, orintravenous.

A pharmaceutical formulation is a composition comprising differentchemical substances including but not limited to active drugs,excipients, etc. which are combined and formulated to produce a finalmedicinal product for the treatment of humans or other organisms.

A sterile formulation is a formulation substantially free of livinggerms or microorganisms.

A therapeutically effective amount is that mass of an active drug in aformulation, and the frequency of administration of a formulation, thatresults in the prevention of the development of symptoms, prevention ofdevelopment of markers or signs of a disease, prevention of thedevelopment of tissue or organ damage, prevention of the progression ofa disease, reduction in the severity of a disease, or treatment ofdisease symptoms as defined above.

Dose range for each individual agent is the range of the mass of activedrug in, and frequency of administration of, a formulation which resultsin the prevention of the development of symptoms, prevention of thedevelopment of a disease, prevention of development of abnormal markersor signs of a disease, prevention of the development of tissue or organdamage, prevention of the progression of a disease, reduction in theseverity of a disease, or treatment of disease symptoms as definedabove.

Regimen means dose, frequency of administration, for example twice-perday, daily, weekly, bi-weekly etc., and duration of treatment, forexample one day, several days, one week, several weeks, one month,several months, one year, several years, etc.

A loading dose is a large initial dose of a substance or series of suchdoses given to more rapidly achieve a therapeutic concentration in thebody. A loading dose can be higher or lower than the maintenance dose.In some instances, therapy is initiated at a loading dose for days,weeks or months in order to rapidly achieve therapeutic levels of thedrug or other chemical entity in tissue, and then the dose is lowered tothe long-term maintenance dose. In some cases, an initial low dose isused for a brief period of time to tolerize and accommodate the patientto the drug (e.g. about 200 mg per day for 1 week), followed by aloading dose (e.g. about 800 mg/day for 12 weeks), followed by themaintenance dose (about 400 mg/day). For hydroxychloroquine sulfate andchloroquine phosphate, the standard dose of 400 mg/day can take 4-6months to achieve therapeutic tissue levels. Therefore, some physiciansuse loading doses of hydroxychloroquine sulfate or chloroquinephosphate, for example a dose of at least about 600 mg/day (about 10mg/kg/day), at least about 800 mg/day (about 13.3 mg/kg/day), at leastabout 1000 mg/day (about 16.67 mg/kg/day), and up to about 1200 mg/day(about 20 mg/kg/d), and up to about 1600 mg/day (about 26.7 mg/kg/day)for 1-16 weeks to more rapidly achieve therapeutic levels in the tissueswhere it is needed for activity. Desethylhydroxychloroquine (DHCQ) isexpected to also accumulate slowly in tissues, such that using, forexample, loading doses of at least about 600 mg/day (about 10mg/kg/day), at least about 800 mg/day (about 13.33 mg/kg/day), at leastabout 1000 mg/day (about 16.67 mg/kg/day), and up to about 1200 mg/day(about 15 mg/kg/d), up to about 1400 mg/day (about 23.33 mg/kg/day), upto about 1600 mg/day (about 26.6 mg/kg/day), for 1-24 weeks, andpreferably for 1-16 weeks may also prove therapeutically beneficial whentreating with DHCQ. Loading doses of HCQ for treatment of inflammatorydisease are discussed in Furst et al. (Arthritis Rheum. 1999 February;42(2):357-65. PMID: 10025931).

Unit doses (also called dosage forms) are essentially pharmaceuticalproducts in the form in which they are marketed for use, typicallyinvolving a mixture of active drug components and nondrug components(excipients), along with other non-reusable material that may not beconsidered either ingredient or packaging (such as a capsule shell, forexample). Depending on the context, multi(ple) unit dose can refer todistinct drug products packaged together, or to a single drug productcontaining multiple drugs and/or doses. The term dosage form can alsosometimes refer only to the chemical formulation of a drug product'sconstituent drug substance(s) and any blends involved.

A dose pack is a premeasured amount of drug to be dispensed to a patientin a set or variable dose and in a package including but not limited toa blister pack or other series of container for the purpose offacilitating a dose regimen. A dose pack can be used to facilitatedelivery of an initial and/or loading dose to an individual, followed bya maintenance dose.

An excipient is generally a pharmacologically inactive substanceformulated with the active pharmaceutical ingredient (“API”) of amedication. Excipients are commonly used to bulk up formulations thatcontain potent active ingredients (thus often referred to as “bulkingagents,” “fillers,” or “diluents”), to allow convenient and accuratedispensation of a drug substance when producing a dosage form. They alsocan serve various therapeutic-enhancing purposes, such as facilitatingdrug absorption or solubility, or other pharmacokinetic considerations.

A “marker of inflammation” (also referred to herein as biomarkers ofinflammation) is an objectively measured characteristic that reflectsthe presence of inflammation in a pre-disease state, or disease stateincluding but not limited to molecular, biochemical, imaging, or grossphysical measurements. As used herein, “markers of inflammation” includeinflammatory markers, metabolic markers, imaging markers, biochemicalmarkers, genetic markers, proteomic markers, gene expression markers,and other markers that can be used to assess inflammation within anindividual. The measurement of abnormal markers in an individualidentifies that individual as being at increased risk for developmentof, in the pre-clinical phases of, in the early-stages of, or having anestablished, inflammatory disease or disease associated withinflammation.

Molecular marker(s) of inflammation (also referred to herein as a“biomarker” or “biomarkers of inflammation”) are molecules obtained fromtissue (e.g., blood) samples of a patient which indicate the presence ofinflammation. Nonlimiting example of such markers can include, forexample, C-reactive protein, a cytokine, an antibody, a DNA sequence, anRNA sequence, a cartilage marker, a metabolic marker, a bone marker, orcombinations thereof. Molecular markers of inflammation includebiochemical markers.

Imaging marker(s) of inflammation (also referred to herein as an“imaging marker” or “imaging biomarkers”) are markers that measure orotherwise determine the presence of inflammation through use of animaging modality, including but not limited to ultrasound, radiography,computerized tomography, magnetic resonance imaging, or nuclear medicalscanning.

Biochemical marker(s) (also referred to herein as a “molecular marker”)are biologic substances that are measured in blood or other tissue as abiomarker. Biological biomarkers of interest include without limitationproteins, nucleic acids, metabolites, fatty acids, peptides, and thelike.

“Inflammatory markers” (also referred to herein as an “inflammatorybiomarkers”) are biomarkers indicating an inflamed state. Inflammatorybiomarkers of interest include without limitation cytokines, chemokines,high sensitivity C-reactive protein (hs-CRP), erythrocyte sedimentationrate (ESR), expression of mRNA encoding inflammatory mediators,inflammatory cells, imaging biomarkers demonstrating inflammation, andother markers indicative of inflammation.

A reference range is defined as the set of values within which 95percent of the normal population falls. It typically refers to the valueor level of a marker (as termed herein as a “marker” or “biomarker”, andexamples of such markers include but are not limited to inflammatorymarkers, metabolic markers, imaging markers, biochemical markers,clinical markers, radiographic markers, and other biomarkers. If thevalue or level of a marker in an individual patient is outside the setof values or levels within which 95 percent of the normal populationfalls, then the marker is considered to exhibit an abnormal level inthat patient (e.g. that patient is determined to have an “abnormalmarker”). In some embodiments, if the value or level of an inflammatorymarker in an individual patient is outside the set of values or levelswithin which 95 percent of the normal population falls, then theinflammatory marker is considered to exhibit an abnormal level in thatpatient (e.g. that patient is determined to have an “abnormalinflammatory marker”). In some embodiments, if the value or level of ametabolic marker (also termed a “metabolic disease marker”) in anindividual patient is outside the set of values or levels within which95 percent of the normal population falls, then the metabolic marker isconsidered to exhibit an abnormal level in that patient (e.g. thatpatient is determined to have an “abnormal metabolic marker”). In someembodiments, if the result of an imaging marker in an individual patientis outside the range of variation of the same imaging marker observedwithin 95 percent of the normal population, then the imaging marker isconsidered to exhibit an abnormal result (e.g. it is an “abnormalimaging marker”). In some embodiments, if the result of a clinicalmarker in an individual patient is outside the range of variation of thesame clinical marker observed within 95 percent of the normalpopulation, then the clinical marker is considered to exhibit anabnormal result (e.g. it is an “abnormal clinical marker”). Themeasurement of abnormal markers in an individual identifies thatindividual as being at increased risk for development of, in thepre-clinical phases of, in the early-stages of, or having anestablished, inflammatory disease or disease associated withinflammation.

Aminoquinolines are derivatives of quinoline, most notable for theirroles as antimalarial drugs. Representative examples of theaminoquinoline class include, but are not limited to 4-aminoquinolines,such as amodiaquine, hydroxychloroquine, chloroquine; and8-aminoquinolines, such as primaquine and pamaquine. Such drugs may beformulated as a “free base” or more usually as a salt thereof.

Metabolites of hydroxychloroquine (HCQ) includedesethylhydroxychloroquine (DHCQ), desethylchloroquine (DHQ), andbisdesethylchloroquine (BDCQ), and their chemical structures arepresented in FIG. 1. FIG. 1B provides a summary of the experimentalfindings presented in FIGS. 2-25 for the anti-inflammatory disease andanti-metabolic disease activity (termed “efficacy”) and the retinaltoxicity (termed “toxicity”) for HCQ and its metabolites DHCQ, DCQ andBDCQ.

The phrase “pharmaceutically acceptable salt(s)”, as used herein, meansthose salts of compounds of the invention that are considered (e.g. byregulatory bodies such as the FDA and EMEA) as safe and effective fororal and topical use in mammals and that possess the desired biologicalactivity. Pharmaceutically acceptable salts include salts of acidic orbasic groups present in compounds of the invention. Pharmaceuticallyacceptable salts include, but are not limited to, hydrochloride,hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acidphosphate, isonicotinate, acetate, lactate, salicylate, citrate,tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate,gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate,p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)), aluminum, calcium,lithium, magnesium, potassium, sodium, zinc, and diethanolamine saltsand the like, as known in the art.

The dose of DHCQ in the present invention comprises, consists of, orconsists essentially of DHCQ or pharmaceutically acceptable salts oresters thereof in a daily dose of at least about 50 mg per day (about0.83 mg/kg/day), at least about 100 mg per day (about 1.67 mg/kg/day),at least about 155 mg per day (about 2.58 mg/kg/day), at least about 200mg per day (about 3.33 mg/kg/day), at least about 250 mg per day (about4.16 mg/kg/day), at least about 300 mg per day (about 5 mg/kg/day), atleast about 310 mg per day (about 5.16 mg/kg/day), at least about 350 mgper day (about 5.83 mg/kg/day), at least about 400 mg per day (about6.67 mg/kg/day), about 450 mg per day (about 7.5 mg/kg/day), about 465mg per day (about 7.75 mg/kg/day), about 500 mg per day (about 8.33mg/kg/day), about 550 mg per day (about 9.16 mg/kg/day), about 600 mgper day (about 10 mg/kg/day), about 620 mg per day (about 10.33mg/kg/day), about 800 mg per day (about 13.33 mg/kg/day), about 930mg/kg/day (about 15.5 mg/kg/day), about 1000 mg per day (about 16.67mg/kg/day), about 1200 mg per day (about 20 mg/kg/day), about 1300 mgper day (about 21.67 mg/kg/day), about 1400 mg per day (about 23.3mg/kg/day), about 1500 mg per day (about 25 mg/kg/day), or about 1600 mgper day (about 26.67 mg/kg/day), inclusive of all ranges and subrangesthere between. In a particular embodiment, the DHCQ pharmaceuticalcomposition is delivered in a once-daily dose.

As discussed herein, in various embodiments, the compositions andmethods of the present invention can comprise any of the enumeratedamounts or daily doses of DHCQ, or pharmaceutically acceptable salts oresters thereof described herein. The DHCQ can be delivered in apharmaceutical composition in the form of a tablet, suspension orcapsule. The DHCQ pharmaceutical composition can be administered inseparate administrations or 2, 3, 4, 5, or 6 equal doses each day.Unless otherwise specified, an indicated dose of a pharmaceuticallyacceptable salt or ester of an active compound, e.g. DHCQ, is expressedas the amount of the corresponding “freebase” (non-salt, non-ester) formof the active compound.

Current recommendations for screening for HCQ-mediated and otheraminoquinoline-mediated retinal toxicity are described (Marmor et al,Ophthalmology. 2011, 118(2):415-22; Bernstein H N. Surv Ophthalmol.October 1967; 12(5):415-47; Anderson C et al, Retina. 2009;29(8):1188-92; Michaelides M et al, Arch Ophthalmol. January 2011;129(1):30-9). The recommendations include performing a baselineexamination of patients starting these drugs to serve as a referencepoint. Annual screening for eye toxicity should begin after 5 years, orsooner if there are additional risk factors including total cumulativedose of more than 1000 g, maintenance dose >6.5 mg/kg/day, renalabnormalities, liver disease, underlying retinal disease, or age greaterthan 60 years of age. Annual screening should include 10-2 automatedfield tests, along with at least one of the following tests: multifocalelectroretinogram (mfERG), spectral domain optical coherence tomography(SD-OCT), or fundus autofluorescence (FAF). Because mfERG testing is anobjective test that evaluates function, it may be used in place ofvisual field tests. Fundus examinations are advised for documentation,but visible bull's-eye maculopathy is a late change, and the goal ofscreening is to detect toxicity at an earlier stage. On annual HCQtoxicity screening examination, demonstration of a worsening of theresults (deterioration of performance on the test and/or worsening ofretinal or macular findings) of the multifocal electroretinogram(mfERG), spectral domain optical coherence tomography (SD-OCT), fundusautofluorescence (FAF), visual field tests, and/or direct visualizationof the macula indicates the development of retinal toxicity. Earlyfundus changes in chloroquine/hydroxychloroquine toxicity include theloss of foveal reflex, macular edema, and pigment mottling that isenhanced with the red-free filter. The appearance of the maculacorrelates poorly with visual-field testing results. Decreased retinaltoxicity (or less retinal toxicity) means that the results of thesetests are stable and unchanged from the baseline results obtained in theinitial pre-treatment baseline exam.

Baseline central visual field examination (perimetry) may be usefulbecause the earliest macular changes due to aminoquinoline and HCQtoxicity are nonspecific and may be indistinguishable from age-relatedchanges. The Humphrey 10-2 program (white target) is recommended forconfirming defects found by the Amsler grid.

Electroretinography (ERG) can be full field, focal, or multifocal. FocalERG techniques can record an ERG response from the foveal and parifovealregions. mfERG, which is typically available in large clinical centers,is more appropriate for the evaluation of chloroquine and/orhydroxychloroquine toxicity because it generates local ERG responsestopographically across the posterior pole and can document a bull's eyedistribution of ERG depression. mfERG objectively evaluates function andcan be used in place of visual fields.

Spectral Domain Optical Coherence Tomography (SD-OCT) measuresperipapillary retinal nerve fiber layer (RNFL) thickness and macularinner and outer retinal thickness in patients with long-term exposure tohydroxychloroquine or chloroquine. OCT is useful to detect peripapillaryRNFL thinning in clinically evident retinopathy. In addition, selectivethinning of the macular inner retina can be detected in the absence ofand before clinically apparent fundus changes.

In animal studies, the first morphologic changes, which become visiblewithin 1 week after initiation of chloroquine treatment, involveganglion cells manifesting membranous cytoplasmic bodies. Other neuralcells of the retina later show these changes. Reversible changes arepresent for up to 5 months of therapy. Prolonged therapy resulted inprogressive degeneration of the ganglion cells and photoreceptor cellbodies and nuclei with outer segment involvement. The most severechanges tended to be perifoveal, with relative foveal sparing.Abnormalities of the pigment epithelium and choroid were seen only afterdegeneration of the ganglion cells and photoreceptors was established.All of the observations described were made before any abnormalitiesbecame detectable in the fundus or on ERG. Pathologic studies ofpatients with chloroquine retinopathy are few and are limited to caseswith advanced retinopathy. Consistent findings include degeneration ofthe outer retina, particularly the photoreceptors and the outer nuclearlayer, with relative sparing of the photoreceptors in the fovea. Pigmentmigration into the retina is seen. Pathologic changes in the ganglioncells have been a consistent finding. Sclerosis of the retinalarterioles is variable.

The use of HCQ is often contraindicated in treating patients atincreased risk for retinal toxicity due to the undesirable safetyprofile of HCQ in such patients. Patients at increased risk for retinaltoxicity include patients with known retinal abnormalities such asdiabetic or hypertensive retinopathy, macular degeneration prior retinaltrauma. There is similar increased risk in any patient with type I ortype II diabetes, patients who have taken HCQ for long periods of time(e.g., about 5 years or more), patients having received cumulative dosesof about 1000 g or more, patients over age 60, or patients of smallstature (ideal body weights of about 60 kg or less) as well as well asobese patients and patients with hepatic or renal impairment.

Withdrawal of the medication and shifting to another form of treatmentis the standard of care for individuals with HCQ and otheraminoquinoline-associated early retinal toxicity or retinalabnormalities. Coordination with the rheumatologist or the dermatologistis warranted for comprehensive care of the patient. If serious toxicsymptoms occur from overdosage or sensitivity, it has been suggestedthat ammonium chloride (8 g daily in divided doses for adults) beadministered orally 3-4 times/wk for several months after therapy hasbeen stopped. Acidification of the urine with ammonium chlorideincreases renal excretion of the 4-aminoquinoline compounds by 20-90%.In patients with impaired renal function and/or metabolic acidosis,caution must be taken.

Recent clinical observations demonstrated that in humans takingconventional doses of HCQ (with 400 mg/day being a common dose), theprevalence of retinal toxicity was 6.8 users per 1,000 (Marmor et al,Ophthalmology. 2011 February; 118(2):415-22). The prevalence wasdependent on the duration of HCQ use. Toxicity sharply increased towards1% after 5-7 years of use. Treatment for >15 years resulted in evenhigher rates of retinal toxicity.

Based on the discoveries presented herein, the rate of retinal toxicityafter long-term treatment of inflammatory diseases or conditions withDHCQ will be lower than that reported for treatment with HCQ (Marmor etal. describes rates of retinal toxicity for treatment with HCQ alone(Ophthalmology. 2011 February; 118(2):415-22)) when the DHCQ and HCQ areused at the same effective total cumulative dose and over the same timeperiod. HCQ-mediated retinal toxicity is identified based on a worseningof the results (deterioration of performance on the test and/orworsening of retinal or macular findings) on annual screening multifocalelectroretinogram (mfERG), spectral domain optical coherence tomography(SD-OCT), fundus autofluorescence (FAF), visual field tests, and/ordirect visualization of the macula. Decreased retinal toxicity (or lessretinal toxicity) means that for a group of individuals treated withDHCQ, there will be at least about a 25%, at least about a 35%, at leastabout a 45%, at least about a 55%, at least about a 65%, at least abouta 75%, and may be around or up to about a 50% lower rate of retinaltoxicity (e.g. toxicity determined based on worsening of function orperformance, or development or worsening of abnormal physicalcharacteristics or findings, of the retina or macula on annual screeningtest results) as compared to that reported for individuals treated withHCQ (or compared to a group of individuals treated with HCQ).

Specific measurements to document the rate (incidence) of retinaltoxicity in individuals receiving treatment with DHCQ as compared toindividuals taking HCQ at a similar effective cumulative dose and over asimilar time period include, without limitation, the following (Marmor,Ophthalmology. 2011 February; 118(2):415-22):

(1) Ophthalmologic Examination. A thorough ophthalmologic dilated fundusexamination to examine the retinal macula for evidence of bull's-eyemaculopathy. Visible bull's-eye retinopathy indicates that toxicity haspersisted long enough to cause RPE degeneration, and is a relativelylate finding. The treatment with DHCQ provides at least about a 25%, atleast about a 35%, at least about a 45%, at least about a 55%, at leastabout a 65%, at least about a 75%, and may be around or up to about a50% lower rate of bull's-eye maculopathy at 5 years, at 10 years, at 15years, and at 20 years of treatment as compared to treatment with HCQ ata similar effective cumulative dose and over a similar time period.

(2) Automated Threshold Visual Fields. Parafoveal loss of visualsensitivity may appear before changes are seen on fundus examination.Automated threshold visual field testing with a white 10-2 pattern(i.e., testing with white targets within 10 degrees of the fovea) giveshigh resolution within the macular region. The finding of anyreproducibly depressed central or parafoveal spots can be indicative ofearly toxicity. Advanced toxicity will typically show a well-developedparacentral scotoma (with or without central sensitivity loss). Thetreatment with DHCQ provides at least about a 25%, at least about a 35%,at least about a 45%, at least about a 55%, at least about a 65%, atleast about a 75%, and up to about a 50% lower rate of depressed centralor parafoveal spots at 5 years, at 10 years, at 15 years, and at 20years of treatment as compared to treatment with HCQ at a similareffective cumulative dose and over a similar time period. The treatmentwith DHCQ is anticipated to be associated with at least about a 25%, atleast about a 35%, at least about a 45%, at least about a 55%, at leastabout a 65%, at least about a 75%, and may be around or up to about a50% lower rate of reproducibly depressed central or parafoveal spots at5 years, at 10 years, at 15 years, and at 20 years of treatment ascompared to treatment with HCQ at a similar effective cumulative doseand over a similar time period.

(3) Spectral Domain-Optical Coherence Tomography. Optical coherencetomography shows a cross-section of retinal layers in the macula.High-resolution instruments (SD or Fourier domain OCT) can showlocalized thinning of the retinal layers in the parafoveal region andconfirm toxicity. Loss of the inner-/outer-segment line may be an earlyobjective sign of parafoveal damage. Further work is needed to evaluatethe sensitivity of SD-OCT relative to visual fields or mfERG, but anumber of cases have shown prominent SD-OCT changes before visual fieldloss; 16, 19-22 SD-OCT testing is rapid and the equipment is availablein many offices and clinics. The treatment with DHCQ provides at leastabout a 25%, at least about a 35%, at least about a 45%, at least abouta 55%, at least about a 65%, at least about a 75%, and up to about a 50%lower rate of localized thinning of the retinal layers in the parafovealregion at 5 years, at 10 years, at 15 years, and at 20 years oftreatment as compared to treatment with HCQ at a similar effectivecumulative dose and over a similar time period.

(4) Fundus Autofluorescence. Autofluorescence imaging may reveal subtleRPE defects with reduced autofluorescence or show areas of earlyphotoreceptor damage (which appear as increased autofluorescence from anaccumulation of outer segment debris). It has the advantage overfluorescein angiography of being faster and not requiring dye injection.Some cases have demonstrated FAF abnormalities before visual field loss.The treatment with DHCQ provides at least about a 25%, at least about a35%, at least about a 45%, at least about a 55%, at least about a 65%,at least about a 75%, and up to about a 50% lower rate of subtle RPEdefects with reduced autofluorescence or areas of early photoreceptordamage at 5 years, at 10 years, at 15 years, and at 20 years oftreatment as compared to treatment with HCQ at a similar effectivecumulative dose and over a similar time period.

(5) Multifocal Electroretinogram. The mfERG generates local ERGresponses topographically across the posterior pole and can objectivelydocument localized paracentral ERG depression in early CQ and HCQretinopathy. mfERG may be more sensitive to early paracentral functionalloss than the white 10-2 field. The treatment with DHCQ provides atleast about a 25%, at least about a 35%, at least about a 45%, at leastabout a 55%, at least about a 65%, at least about a 75%, and up to abouta 50% lower rate of localized paracentral ERG depression at 5 years, at10 years, at 15 years, and at 20 years of treatment as compared totreatment with HCQ at a similar effective cumulative dose and over asimilar time period.

As described herein, as compared to the rates of retinal toxicitydescribed in the art, given a similar level of therapeutic activity andtime period of dosing for HCQ, treatment with DHCQ provides less retinaltoxicity as compared to treatment with HCQ used at a similar effectivetotal cumulative HCQ dose. In one embodiment, substantially withoutretinal toxicity means that in a patient population analogous to thatdescribed by the American College of Opthamolology and Marmor et al(Ophthalmology. 2011 February; 118(2):415-22), in which the retinaltoxicity rate approached 1% after 5 years in individuals treated withHCQ, treatment with DHCQ will reduce the rate of retinal toxicity toless that about 0.5% of treated individuals. Retinal toxicity isidentified based on worsening of the results (deterioration ofperformance on the test, and/or development or worsening of physicalabnormalities, of retinal or macular physical characteristics orfindings) on annual screening multifocal electroretinogram (mfERG),spectral domain optical coherence tomography (SD-OCT), fundusautofluorescence (FAF), visual field tests, and/or direct visualizationof the macula examinations. Further, given current assumptions thatretinal screening is justified as rates of toxicity approach 1%, thereduction in cumulative rates of retinal toxicity associated with DHCQtreatment can in turn reduce the need for retinal toxicity screening toa single screening at 5 and 10 years, or to entirely negate the need forscreening, or to performing screening on an every other year basisstarting 7 years following initiation of therapy. Due to the lowerretinal toxicity of DHCQ, use of DHCQ will enable a higher totalcumulative dose to be delivered as compared to treatment with HCQ,thereby enabling dosing of DHCQ at higher daily doses and/or over alonger period of time which to provide greater efficacy in treating theinflammatory disease. Due to the lower retinal toxicity of DHCQ, use ofDHCQ will enable treatment of individuals at increased risk, in thepre-clinical phases, or in the early-stages of an inflammatory diseaseor disease associated with inflammation who require treatment over along period of time to prevent development of the inflammatory diseaseor disease associated with inflammation.

In another embodiment, substantially without retinal toxicity means thatin groups of subjects in which one group is treated with DHCQ and asecond group is treated with HCQ (when the DCHQ and HCQ are used at thesame total cumulative doses), that after 5 years of treatment the grouptreated with DHCQ will exhibit an approximately 50% lower incidence ofretinal toxicity as compared to the group treated with HCQ. In anotherembodiment, substantially without retinal toxicity means that in groupsof subjects in which one group is treated with DHCQ and a second groupis treated with HCQ (when the DHCQ and HCQ are used at the same totalcumulative dose), that after 10 years of treatment the group treatedwith DHCQ will exhibit an approximately 50% lower incidence of retinaltoxicity as compared to the group treated with HCQ. In anotherembodiment, substantially without retinal toxicity means that in groupsof subjects in which one group is treated with DHCQ and a second groupis treated with HCQ (when the DHCQ and HCQ are used at the same totalcumulative dose), that after 15 years of treatment the group treatedwith DHCQ will exhibit an approximately 50% lower retinal toxicity ascompared to the group treated with HCQ. In another embodiment,substantially without retinal toxicity means that in groups of subjectsin which one group is treated with DHCQ and a second group is treatedwith HCQ (when the DHCQ and HCQ are used at a similar total cumulativedoses), that after 20 years of treatment the group treated with DHCQwill exhibit an approximately 50% lower incidence of retinal toxicity ascompared to the group treated with HCQ.

In addition to reducing the incidence of retinal toxicity as describedabove, the use of DHCQ can reduce the severity of retinal toxicity whenit does occur. The use of DHCQ reducing the severity of retinal toxicitymeans that for an individual taking DHCQ that develops retinal toxicitythat there will be at least about a 25%, at least about a 35%, at leastabout a 45%, at least about a 55%, at least about a 65%, at least abouta 75%, and may be around or up to about a 50% reduction in the severityof the retinal toxicity (e.g. the degree of toxicity determined based onworsening of function or performance, or development or worsening ofabnormal physical characteristics or findings, of the retina or maculaon annual screening test results) as compared to that reported forindividuals treated with a similar cumulative dose of, and over asimilar time frame with, HCQ.

As discussed above, current recommendations for screening forHCQ-mediated and other aminoquinoline-mediated retinal toxicity aredescribed (Marmor et al, Ophthalmology. 2011, 118(2):415-22; Bernstein HN. Surv Ophthalmol. October 1967; 12(5):415-47; Anderson C et al,Retina. 2009; 29(8):1188-92; Michaelides M et al, Arch Ophthalmol.January 2011; 129(1):30-9). The recommendations include performing abaseline examination of patients starting these drugs to serve as areference point. Following this baseline exam, the recommendations arefor annual screening for retinal toxicity begin at 5 years of HCQtreatment in individuals who do not have risk factors for retinaltoxicity. For individuals with risk factors for retinal toxicity, it isrecommended that annual screening begin before an individual reaches 5years of HCQ therapy. The recommendations outline risk factors forretinal toxicity as including one or more of the following: totalcumulative dose of HCQ sulfate of more than 1000 g, maintenance dose ofHCQ sulfate >6.5 mg/kg/day, renal insufficiency, liver disease,underlying retinal disease, age older than 60 years of age. Annualscreening should include 10-2 automated field tests, along with at leastone of the following tests: multifocal electroretinogram (mfERG),spectral domain optical coherence tomography (SD-OCT), or fundusautofluorescence (FAF). Because mfERG testing is an objective test thatevaluates function, it may be used in place of visual field tests.Fundus examinations are advised for documentation, however, visiblebull's-eye maculopathy is a late change, and the goal of screening is todetect toxicity at an earlier stage. On annual HCQ toxicity screeningexamination, demonstration of a worsening of the results (deteriorationof performance on the test and/or worsening of retinal or macularfindings) of the multifocal electroretinogram (mfERG), spectral domainoptical coherence tomography (SD-OCT), fundus autofluorescence (FAF),visual field tests, and/or direct visualization of the macula indicatesthe development of retinal toxicity. Early fundus changes inchloroquine/hydroxychloroquine toxicity include the loss of fovealreflex, macular edema, and pigment mottling that is enhanced with thered-free filter. The appearance of the macula correlates poorly withvisual-field testing results. Decreased retinal toxicity (or lessretinal toxicity) means that the results of these tests are stable andunchanged from the baseline results obtained in the initialpre-treatment baseline exam.

The discovery described herein of lower toxicity of DHCQ relative toconventional therapies such as HCQ reduces the concerns regardingretinal toxicity, and therefore retinal toxicity screening can be safelyinitiated much later when DHCQ is used, compared to HCQ. In oneembodiment, the use of DHCQ would allow retinal toxicity screening to besafely performed beginning at about 5 years following initiation oftherapy. In another embodiment, the use of DHCQ would allow retinaltoxicity screening to be safely performed beginning at about 7 yearsfollowing initiation of therapy. In another embodiment, the use of DHCQwould allow retinal toxicity screening to be safely performed beginningat about 10 years following initiation of therapy. In anotherembodiment, the use of DHCQ would allow retinal toxicity screening to besafely performed beginning at about 15 years following initiation oftherapy. In another embodiment, the use of DHCQ would allow retinaltoxicity screening to be safely performed beginning at about 20 yearsfollowing the initiation of therapy. In another embodiment, the use ofDHCQ would allow for no retinal toxicity screening. In contrast, the useof HCQ requires retinal toxicity screening at baseline and annuallystarting 5 years following the initiation of HCQ therapy.

Similarly, the lower toxicity of DHCQ permits less frequent retinaltoxicity screening compared to treatment with HCQ. In one embodiment,the use of DHCQ allows retinal toxicity screening to be safely performedat about 1 year intervals. In another embodiment, the use of DHCQ allowsretinal toxicity screening to be safely performed at about 18 monthintervals. In another embodiment, the use of DHCQ allows retinaltoxicity screening to be safely performed at about 2 year intervals. Inanother embodiment, the use of DHCQ allows retinal toxicity screening tobe safely performed at about 3 year intervals. In another embodiment,the use of DHCQ allows retinal toxicity screening to be safely performedat about 5 year intervals. In another embodiment, the use of DHCQ allowretinal toxicity screening to be safely performed at about 7 yearintervals. In another embodiment, the use of DHCQ allows retinaltoxicity screening to be safely performed at about 10 year intervals. Inanother embodiment, the use of DHCQ enables a patient to continuetreatment without any retinal toxicity screening.

In another embodiment, the therapeutic use of DHCQ allows the safe useor continued use of aminoquinoline therapy in those at increased riskfor retinal toxicity, including but not limited to individuals withprior use of hydroxychloroquine. In one embodiment, the use of DHCQenables the treatment (or continued treatment) of individuals who are atrisk of unacceptable levels of retinal toxicity because they previouslyused HCQ for longer than about 2.5 years. In another embodiment, the useof DHCQ enables the treatment (or continued treatment) of individualswho are at risk of unacceptable levels of retinal toxicity because theypreviously used HCQ for longer than about 5 years. In anotherembodiment, the use of DHCQ enables the treatment (or continuedtreatment) of individuals who are at risk of unacceptable levels ofretinal toxicity because they previously used HCQ for longer than about7 years. In another embodiment, the use of DHCQ enables the treatment(or continued treatment) of individuals who are at risk of unacceptablelevels of retinal toxicity because they previously used HCQ for longerthan about 10 years. In another embodiment, the use of DHCQ enables thetreatment (or continued treatment) of individuals who are at risk ofunacceptable levels of retinal toxicity because they previously used HCQfor longer than about 15 years.

In another embodiment, the use of DHCQ therapy enables aminoquinolinetherapy (or continued therapy) in individuals over age >60 years. Inanother embodiment, the use of DHCQ therapy enables treatment ofindividuals with small stature or low body weight, with retinopathy, atrisk for underlying retinopathy, other ocular pathology, or have taken1000 grams or more of HCQ. Short stature, in certain cases referred toas small stature or diminutive stature in the literature, refers tohumans who weight <60 Kg or less that 135 lbs.

In another embodiment, the use of DHCQ allows the use of aminoquinolinetherapy (or continued therapy) in patients who are at risk ofunacceptable levels of retinal toxicity because they have taken doses ofHCQ of 1000 grams or more in their lifetime. In another embodiment, theuse of DHCQ allows the use of aminoquinoline therapy (or continuedtherapy) in patients who are at risk of unacceptable levels of retinaltoxicity because they have taken doses of HCQ of 2000 grams or more intheir lifetime. In another embodiment, the use of DHCQ allows the use ofaminoquinoline therapy (or continued therapy) in patients who are atrisk of unacceptable levels of retinal toxicity because they have takendoses of HCQ of 3000 grams or more in their lifetime. In anotherembodiment, the use of DHCQ allows the use of aminoquinoline therapy (orcontinued therapy) in patients who are at risk of unacceptable levels ofretinal toxicity because they have taken doses of HCQ of 5000 grams ormore in their lifetime. In another embodiment, the use of DHCQ reducesthe risk of aminoquinoline therapy in patients who are at risk ofunacceptable levels of retinal toxicity because they have taken doses ofHCQ >500 grams in their lifetime. In another embodiment, the use of DHCQreduces the risk of aminoquinoline therapy in patients who are at riskof unacceptable levels of retinal toxicity because they have taken dosesof HCQ >250 grams in their lifetime. In another embodiment, the use ofDHCQ reduces the risk of aminoquinoline therapy in patients who are atrisk of unacceptable levels of retinal toxicity because they have takendoses of HCQ >100 grams in their lifetime. In another embodiment, theuse of DHCQ reduces the risk of aminoquinoline therapy in patients whoare at risk of unacceptable levels of retinal toxicity because they havetaken doses of HCQ >50 grams in their lifetime.

In another embodiment, the use of DHCQ allows the safe use ofaminoquinoline therapy in patients who are at risk of unacceptablelevels of retinal toxicity because they have taken doses of DHCQ of 1000grams or more in their lifetime. In another embodiment, the use of DHCQallows the safe use of aminoquinoline therapy in patients who are atrisk of unacceptable levels of retinal toxicity because they have takendoses of DHCQ of 2000 grams or more in their lifetime. In anotherembodiment, the use of DHCQ allows the safe use of aminoquinolinetherapy in patients who have taken doses of DHCQ of 3000 grams or morein their lifetime. In another embodiment, the use of DHCQ allows thesafe use of aminoquinoline therapy in patients who have taken doses ofDHCQ of 5000 grams or more in their lifetime. In another embodiment, theuse of DHCQ provides a reduced risk of using of aminoquinoline therapyin patients who have taken doses of DHCQ >500 grams in their lifetime.In another embodiment, the use of DHCQ provides a reduced risk of usingof aminoquinoline therapy in patients who have taken doses of DHCQ >250grams in their lifetime. In another embodiment, the use of DHCQ providesa reduced risk of using of aminoquinoline therapy in patients who havetaken doses of DHCQ >100 grams in their lifetime. In another embodiment,the use of DHCQ provides a reduced risk of using of aminoquinolinetherapy in patients who have taken doses of DHCQ >50 grams in theirlifetime.

In another embodiment, the use of DHCQ provides a reduced risk ofretinal toxicity from aminoquinoline therapy in patients that would becandidates for therapy with antimalarials including but not limited toHCQ. In another embodiment, the use of DHCQ provides a reduced risk ofretinal toxicity from aminoquinoline therapy in obese patients (bodymass index >30) that would be candidates for therapy with anti-malarialsincluding but not limited to HCQ. In another embodiment, the use of DHCQprovides a reduced risk of retinal toxicity from aminoquinoline therapyin patients or small stature (body mass index <18.5) and/or low idealbody weight (<60 Kg) that would be candidates for therapy withanti-malarials including but not limited to hydroxychloroquine.

In another embodiment, the use of DHCQ provides a reduced risk ofretinal toxicity from aminoquinoline therapy in patients with renalimpairment (creatinine clearance <60 ml/ml) or hepatic impairment (serumalbumin <3.5 mg/dL or INR >1.2 or direct bilirubin >0.2 mg/dL) thatwould be candidates for therapy with anti-malarials including but notlimited to HCQ.

In another embodiment, the use of DHCQ provides a reduced risk of usingof aminoquinoline therapy in patients with underlying retinal disease(including diabetic or hypertensive retinopathy, macular degeneration,or prior retinal trauma) that would be candidates for therapy withantimalarials including but not limited to hydroxychloroquine

In another embodiment use of DHCQ provides a reduced risk of using ofaminoquinoline therapy in patients at risk for concurrent retinaldisease (including diabetic patients, hypertensive patients, andpatients with a strong family history of macular degeneration) thatwould be candidates for therapy with antimalarials including but notlimited to hydroxychloroquine

Statins are inhibitors of HMG-CoA reductase enzyme. These agents aredescribed in detail in various publications. For example, mevastatin andrelated compounds are disclosed in U.S. Pat. No. 3,983,140, lovastatin(mevinolin) and related compounds are disclosed in U.S. Pat. No.4,231,938, pravastatin and related compounds are disclosed in U.S. Pat.No. 4,346,227, simvastatin and related compounds are disclosed in U.S.Pat. Nos. 4,448,784 and 4,450,171; fluvastatin and related compounds aredisclosed in U.S. Pat. No. 5,354,772; atorvastatin and related compoundsare disclosed in U.S. Pat. Nos. 4,681,893, 5,273,995 and 5,969,156; andcerivastatin and related compounds are disclosed in U.S. Pat. Nos.5,006,530 and 5,177,080. Additional statin compounds are disclosed inU.S. Pat. Nos. 5,208,258, 5,130,306, 5,116,870, 5,049,696, RE 36,481,and RE 36,520. Statins include the salts and/or ester thereof.

For the purposes of the present invention, an effective dose of a statinin a combination with DHCQ (or salt or ester thereof) is the dose that,when administered for a suitable period of time, usually at least aboutone week, about two weeks or more, or up to extended periods of timesuch as months or years, will evidence a reduction in the progression ofthe disease. It will be understood by those of skill in the art that aninitial dose may be administered for such periods of time, followed bymaintenance doses, which, in some cases, will be at a reduced dosage.

The formulation and administration of statins is well known, and willgenerally follow conventional usage. The dosage required to treatinflammation may be commensurate with the dose used in the treatment ofhigh cholesterol. For example, atorvastatin may be administered in adaily dose of at least about 1 mg, at least about 5 mg, at least about10 mg, and not more than about 250 mg, not more than about 150 mg, ornot more than about 80 mg, inclusive of a values, ranges, and subrangestherebetween. The use of statins in general and atorvastatin inparticular can be at doses from about 1-250 mg (about 0.01-2.5 mg/kg),and DHCQ specifically from about 50-1000 mg per day (about 0.83-16.67mg/kg). Table 1 presents the half life (T_(1/2)), maximum concentration(C_(max) in mg/L), the time it takes to reach C_(max) (T_(max) inhours), volume distribution (V_(d) in L) and percent oralbioavailability of atorvastatin.

The DHCQ (or salt or ester thereof), and/or statins can be incorporatedinto a variety of formulations for therapeutic administration. Moreparticularly, the compounds of the present invention can be formulatedinto pharmaceutical compositions by combination with appropriatepharmaceutically acceptable carriers or diluents either alone or incombination with an aminoquinoline, and may be formulated intopreparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.As such, administration of the compounds can be achieved in variousways, including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, intraarticular, etc.,administration. The active agent may be systemic after administration ormay be localized by the use of regional administration, intramuraladministration, or use of an implant that acts to retain the active doseat the site of implantation. In particular embodiments, the formulationsare oral formulations.

The use of combination therapy may allow lower doses of each monotherapythan currently used in standard practice while achieving significantefficacy, including efficacy beyond that conventional dosing of eithermonotherapy. Those of skill in the art will readily appreciate that doselevels can vary as a function of the specific compound, the severity ofthe symptoms, and the susceptibility of the subject to side effects.Some of the specific compounds are more potent than others. Preferreddosages for a given compound are readily determinable by those of skillin the art by a variety of means. A preferred means is to measure thephysiological potency of a given compound. The use of combinationtherapy may allow lower doses of each monotherapy than currently used instandard practice while achieving significant efficacy, includingefficacy greater than that achieved by conventional dosing of eithermonotherapy.

Specific examples of statins useful in the methods of the invention areatorvastatin (LIPITOR™); cerivastatin (LIPOBAY™); fluvastatin (LESCOL™);lovastatin (MEVACOR™); mevastatin (COMPACTIN™); pitavastatin (LIVALO™);pravastatin (PRAVACHOL™); Rosuvastatin (CRESTOR™); simvastatin (ZOCOR™);etc.

A combination drug product of the invention, which can be provided as asingle formulation or as two separate formulations of the activeingredients, DHCQ and a statin. In particular embodiments thecombination provides for a synergistic improvement in disease markers ordisease symptoms over the administration of either drug as a singleagent.

In some embodiments, the formulation or combination of active agentsconsists essentially of the combination of DHCQ and atorvastatin, i.e.no additional active agents are included in the formulation, althoughexcipients, packaging and the like will be present. In some embodimentsthe formulation is free of NSAIDs, including aspirin. In someembodiments the formulation is free of folic acid or folate.Importantly, this combination does not require use of an antibiotic, ananti-viral, or an anti-bacterial agent, and in some embodiments theformulation is free of antibiotics, anti-viral, or anti-bacterialagents.

The combination can be defined based on the dose ratio of the two drugs,where the DHCQ is usually expressed as the amount of base drug that ispresent, i.e. not including the weight contribution of the counter ion.Where the composition comprises DHCQ and atorvastatin, the ratio ofthese two active agents may range from about 160 mg:80 mg (about 2.6mg/kg:1.3 mg/kg) to about 600 mg:1 mg (about 10 mg/kg:0.016 mg/kg), fromabout 500 mg:100 mg (about 8.33 mg/kg:1.6 mg/kg) to about 500 mg:10 mg(about 8.33 mg/kg:0.16 mg/kg), from about 100 mg:10 mg (about 1.6mg/kg:0.16 mg/kg) to about 600 mg:10 mg (about 10 mg/kg:0.16 mg/kg), toabout 150 mg:10 mg (2.5 mg/kg:0.16 mg/kg), to about 600 mg:20 mg (about10 mg/kg:0.28 mg/kg).

In a particular embodiment, the combination ofdesethylhydroxychloroquine (DHCQ) and atorvastatin are administered inone of the following once-daily fixed dosages (DHCQ base mg:atorvastatinbase mg): about 800:80, about 600:80, about 500:80, about 465:80, about450:80, about 425:80, about 400:80, about 375:80, about 325:80, about310:80, about 300:80, about 275:80, about 250:80, about 225:80, about200:80, about 155:80 about 100:80, about 800:60, about 600:60, about500:60, about 465:60, 450:60, about 425:60, about 400:60, about 375:60,about 325:60, about 310:60, about 300:60, about 275:60, about 250:60,about 225:60, about 200:60, about 155:60 about 100:60, 800:50, 600:50,500:50, 465:50, 450:50, 425:50, 400:50, 375:50, 325:50, 310:50, about300:50, about 275:50, about 250:50, about 225:50, about 200:50, about155:50, about 100:50, about 800:45, about 600:45, about 500:45, about465:45, about 450:45, about 425:45, about 400:45, about 375:45, about325:45, about 310:45, about 300:45, about 275:45, about 250:45, about225:45, about 200:45, about 155:45, about 100:45, about 800:40, about600:40, about 500:40, about 465:40, about 450:40, about 425:40, about400:40, about 375:40, about 325:40, about 310:40, about 300:40, about275:40, about 250:40, about 225:40, about 200:40, about 155:40, about100:40, about 800:35, about 600:35, about 500:35, about 465:35, about450:35, about 425:35, about 400:35, about 375:35, about 325:35, about310:35, about 300:35, about 275:35, about 250:35, about 225:35, about200:35, about 155:35, about 100:35, about 800:30, about 600:30, about500:30, about 465:30, about 450:30, about 425:30, about 400:30, about375:30, about 325:30, about 310:30, about 300:30, about 275:30, about250:30, about 225:30, about 200:30, about 155:30, about 100:30, about800:25, about 600:25, about 500:25, about 465:25, about 450:25, about425:25, about 400:25, about 375:25, about 325:25, about 310:25, about300:25, about 275:25, about 250:25, about 225:25, about 200:25, about155:25, about 100:25, about 800:20, about 600:20, about 500:20, about465:20, about 450:20, about 425:20, about 400:20, about 375:20, about325:20, about 310:20, about 300:20, about 275:20, about 250:20, about225:20, about 200:20, about 155:20, about 100:20, about 800:15, about600:15, about 500:15, about 465:15, about 450:15, about 425:15, about400:15, about 375:15, about 325:15, about 310:15, about 300:15, about275:15, about 250:15, about 225:15, about 200:15, about 155:15, about100:15, about 800:10, about 600:10, about 500:10, about 465:10, about450:10, about 425:10, about 400:10, about 375:10, about 325:10, about310:10, about 300:10, about 275:10, about 250:10, about 225:10, about200:10, about 155:10, about 100:10, about 800:5, about 600:5, about500:5, about 465:5, about 450:5, about 425:5, about 400:5, 375:5, about325:5, about 310:5, about 300:5, about 275:5, about 250:5, about 225:5,200:5, about 155:5, or about 100:5.

For demonstrating the synergistic activity of the two drugs (e.g., DHCQand a statin such as atorvastatin) and establishing an appropriatefixed-dose ratio for clinical investigation, varying amounts of the twodrugs are administered to appropriate animal models of inflammatorydisease, either at a time of active disease (following disease onset) orat an early time point representative of pre-clinical disease, and theeffect on disease activity or progression is measured. Alternatively,the effects of varying amounts of the two drugs are tested on a cellularresponse mediating inflammation that may be involved in the pathogenesisof disease.

It is within the level of skill of a clinician to determine thepreferred route of administration and the corresponding dosage form andamount, as well as the dosing regimen, i.e., the frequency of dosing. Inparticular embodiments, the combination therapy will be delivered inonce-a-day (s.i.d.) dosing. In other embodiments, twice-a-day (b.i.d.)dosing may be used. However, this generalization does not take intoaccount such important variables as the specific type of inflammatorydisease, the specific therapeutic agent involved and its pharmacokineticprofile, and the specific individual involved. For an approved productin the marketplace, much of this information is already provided by theresults of clinical studies carried out to obtain such approval. Inother cases, such information may be obtained in a straightforwardmanner in accordance with the teachings and guidelines contained in theinstant specification taken in light of the knowledge and skill of theartisan. The results that are obtained can also be correlated with datafrom corresponding evaluations of an approved product in the sameassays.

In some embodiments, the DHCQ is dosed at a higher initial dosing range(dose loading) to ensure more rapid achievement of therapeutic levels inblood and tissue, because this agent is known to have wide distributionand thus an extended terminal half-life. Such loading achievessteady-state blood levels, and increases tissue levels, more rapidlythan single-dose daily dosing and results in earlier therapeuticefficacy (Furst et al, Arthritis Rheum. 1999 February; 42(2):357-65).Based on the loading dose used for HCQ, the typical dose loading may bein the range of about 500-1600 mg/d (about 8.33-26.6 mg/kg/d) for about1-24 weeks, or for about 1-16 weeks, for DHCQ. This dose loading is doneeither alone, administered separately from the statin, or combined withthe statin, including use of a “dose pack” with a blister packaging orother mechanism that provides clear information about daily dosing thatwould facilitate initial dose loading followed by continuation with astable daily dosing, or other regular dosing intervals sufficient toachieve target drug levels and pharmacodymamic efficacies. The loadingdose is typically delivered daily for 1-16 weeks, following which thedose is decreased to the typical maintenance dose of about 400-800 mgper day (about 6.67-13.3 mg/kg/day), or about 550-700 mg per day(9.16-11.67 mg/kg/day). DHCQ can be delivered in once-daily doses (e.g.about 600 mg per day orally [about 10 mg/kg/day]), or in a dividedtwice-daily dose (e.g. about 300 mg orally twice per day [for a total ofabout 10 mg/kg/day]). DHCQ can be delivered in once-daily doses (e.g.about 550 mg per day orally [about 9.16 mg/kg/day]), or in a dividedtwice-daily dose (e.g. about 275 mg orally twice per day [for a total ofabout 9.16 mg/kg/day]).

In one aspect, the present invention provides a unit dosage form of theformulation of the invention. The term “unit dose” or “unit dosageform,” refers to physically discrete units suitable as unitary dosagesfor human subjects, each unit containing a predetermined quantity ofdrugs in an amount calculated sufficient to produce the desired effectin association with a pharmaceutically acceptable diluent, carrier, orvehicle. The specifications for the unit dosage forms of the presentinvention depend on the particular combination employed and the effectto be achieved, and the pharmacodynamics associated with the host.

In one aspect, the DHCQ is formulated into a pharmaceutical compositionby combination with appropriate, pharmaceutically acceptable carriers ordiluents, and are formulated into preparations in solid, semi-solid,liquid, suspension, emulsion, or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suspensions,emulsions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration can be achieved in various ways,usually by oral administration. In pharmaceutical dosage forms, thedrugs may be administered in the form of their pharmaceuticallyacceptable salts, or they may also be used alone or in appropriateassociation, as well as in combination with other pharmaceuticallyactive compounds. The following methods and excipients are exemplary andare not to be construed as limiting the invention.

In particular embodiments, the T_(max) of DHCQ after administration ofthe compositions of the present invention ranges from about 15 minutesto about 2 hours, about 15 minutes to about 1 hour, about 30 minutes toabout 1 hour, about 30 minutes to about 2 hours, about 30 minutes toabout 3 hours, about 1 hour to about 2 hours, about 1 hour to about 2.5hours, about 1 hour to about 3 hours, about 1 hour to about 4 hours, orabout 1 hour to about 5 hours, about 2 hours to about 6 hours, about 2hours to about 7 hours, about 1 hour to about 8 hours, about 1 hour toabout 9 hours, about one hour to about 10 hours, about 1 hour to about11 hours, about 1 hour to about 12 hours. In some other embodiments, theT_(max) of DHCQ after administration of the compositions of the presentinvention ranges from about 2 hours to 2.5 hours, about 2-3 hours, about2-4 hours, about 2-5 hours, about 2-6 hours, about 2-7 hours, about 2-8hours, about 2-9 hours, about 2-10 hours, about 2-11 hours, or about2-12 hours.

In particular embodiments, the C_(max) (in ng/ml of whole blood) of DHCQafter administration of the compositions of the present invention rangesfrom about 25-50, about 25-100, about 25-150, about 25-200, about 50-75,about 50-100, about 50-150, about 50-200, about 50-250, about 50-300,about 50-400, or about 50-500. In another embodiment, the C_(max) (inng/ml of whole blood) of DHCQ after administration of the compositionsof the present invention ranges from about 100-150, about 100-200, about100-250, about 100-250, about 100-300, about 100-350, about 100-400,about 100-450, about 100-500, about 100-550, about 100-600, or about100-700. In another embodiment, the C_(max) (in ng/ml of whole blood) ofDHCQ after administration of the compositions of the present inventionranges from about 150-200, about 150-250, about 150-300, about 150-350,about 150-400, about 150-450, about 150-500, about 150-550, about150-600, about 150-650, about 150-700, or about 150-800. In anotherembodiment, the C_(max) (in ng/ml of whole blood) of DHCQ afteradministration of the compositions of the present invention ranges fromabout 200-250, about 200-300, about 200-350, about 200-400, about200-450, about 200-500, about 200-550, about 200-600, about 200-650,about 200-700, about 200-750, or about 200-800. In another embodiment,the C_(max) (in ng/ml of whole blood) of DHCQ after administration ofthe compositions of the present invention ranges from about 250-300,about 250-350, about 250-400, about 250-450, about 250-500, about250-550, about 250-600, about 250-650, about 250-700, about 250-750,about 250-800, or about 250-850.

In particular embodiments, the volume of distribution (V_(d); in liters)of DHCQ after administration of the compositions of the presentinvention ranges from about 100-150, about 100-200, about 100-250, about100-250, about 100-300, about 100-350, about 100-400, about 100-450,about 100-500, about 100-550, about 100-600, or about 100-700. Inanother embodiment, the volume of distribution (V_(d); in liters) ofDHCQ after administration of the compositions of the present inventionranges from about 150-200, about 150-250, about 150-300, about 150-350,about 150-400, about 150-450, about 150-500, about 150-550, about150-600, about 150-650, about 150-700, or about 150-800. In anotherembodiment, the volume of distribution (V_(d); in liters) of DHCQ afteradministration of the compositions of the present invention ranges fromabout 200-250, about 200-300, about 200-350, about 200-400, about200-450, about 200-500, about 200-550, about 200-600, about 200-650,about 200-700, about 200-750, or about 200-800. In another embodiment,the volume of distribution (V_(d); in liters) of DHCQ afteradministration of the compositions of the present invention ranges fromabout 250-300, about 250-350, about 250-400, about 250-450, about250-500, about 250-550, about 250-600, about 250-650, about 250-700,about 250-750, about 250-800, or about 250-850. In another embodiment,the volume of distribution (V_(d); in liters) of DHCQ afteradministration of the compositions of the present invention ranges fromabout 300-350, about 300-400, about 300-450, about 300-350, about300-400, about 300-450, about 300-500, about 300-550, about 300-600,about 300-650, about 300-700, about 300-750. In another embodiment, thevolume of distribution (V_(d); in liters) of DHCQ after administrationof the compositions of the present invention ranges from about 350-400,about 350-450, about 350-500, about 350-550, about 350-600, about350-650, about 350-700, about 350-750, about 350-800, about 350-850,about 350-900, about 350-950. In another embodiment, the volume ofdistribution (V_(d); in liters) of DHCQ after administration of thecompositions of the present invention ranges from about 400-450, about400-500, about 400-550, about 400-600, about 400-650, about 400-700,about 400-750, about 400-800, about 400-850, about 400-900, about400-950, or about 400-1000.

In particular embodiments, the clearance (CI; in liters per hour) ofDHCQ after administration of the compositions of the present inventionranges from about 0.5-1, about 0.5-1.5, about 0.5-2, about 0.5-2.5,about 0.5-3, about 0.5-3.5, about 0.5-4, about 0.5-4.5, about 0.5-5,about 0.5-5.5, about 0.5-6, about 0.5-6.5. In another embodiment, theclearance (CI; in liters per hour) of DHCQ after administration of thecompositions of the present invention ranges from about 1-1.5, about1-2, about 1-2.5, about 1-3, about 1-3.5, about 1-4, about 1-4.5, about1-5, about 1-5.5, about 1-6, about 1-6.5, about 1-7. In anotherembodiment, the clearance (CI; in liters per hour) of DHCQ afteradministration of the compositions of the present invention ranges fromabout 1.5-2, about 1.5-2.5, about 1.5-3, about 1.5-3.5, about 1.5-4,about 1.5-4.5, about 1.5-5, about 1.5-5.5, about 1.5-6, about 1.5-6.5,about 1.5-7, about 1.5-7.5. In another embodiment, the clearance (CI; inml per hour) of DHCQ after administration of the compositions of thepresent invention ranges from about 150-200, about 150-250, about150-300, about 150-350, about 150-400, about 150-450, about 150-500,about 150-550, about 150-600, about 150-650, about 150-700, or about150-800.

In another embodiment, the clearance (CI; in ml per hour) of DHCQ afteradministration of the compositions of the present invention ranges fromabout 250-300, about 250-350, about 250-400, about 250-450, about250-500, about 250-550, about 250-600, about 250-650, about 250-700,about 250-750, about 250-800, or about 250-850. In another embodiment,the clearance (CI; in ml per hour) of DHCQ after administration of thecompositions of the present invention ranges from about 350-400, about350-450, about 350-500, about 350-550, about 350-600, about 350-650,about 350-700, about 350-750, about 350-800, about 350-850, about350-900, or about 350-950. In another embodiment, the clearance (CI; inml per hour) of DHCQ after administration of the compositions of thepresent invention ranges from about 400-450, about 400-500, about400-550, about 400-600, about 400-650, about 400-700, about 400-750,about 400-800, about 400-850, about 400-900, about 400-950, or about400-1000.

For oral preparations, DHCQ can be used alone or in combination withappropriate additives to make tablets, suspensions, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch, or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch, orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives, and flavoring agents.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are commercially available. Moreover,pharmaceutically acceptable auxiliary substances, such as pH-adjustingand buffering agents, tonicity-adjusting agents, stabilizers, wettingagents and the like, are commercially available. Any compound useful inthe methods and compositions of the invention can be provided as apharmaceutically acceptable base-addition salt. “Pharmaceuticallyacceptable base-addition salt” refers to those salts that retain thebiological effectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared byadding an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Preferred inorganicsalts are the ammonium, sodium, potassium, calcium, and magnesium salts.Salts derived from organic bases include, but are not limited to, saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like. Exemplaryorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline, and caffeine. In someembodiments the salt is chloride or sulfate. In other embodiments thesalt is a bidentate salt such as fumaric acid or succinic acid.

The active agent, or their pharmaceutically acceptable salts may containone or more asymmetric centers and may thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that may be defined, interms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or(L)-for amino acids. The present invention is meant to include all suchpossible isomers, as well as, their racemic and optically pure forms.Optically active (+) and (−), (R)- and (S)-, or (D)- and (L)-isomers maybe prepared using chiral synthons or chiral reagents, or resolved usingconventional techniques, such as reverse phase HPLC. When the compoundsdescribed herein contain olefinic double bonds or other centers ofgeometric asymmetry, and unless specified otherwise, it is intended thatthe compounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

The active agents of the present invention or salts thereof may form asolvate and/or a crystal polymorph, and the present invention containssuch solvates and crystal polymorphs of various types. A solvate means asolvate of the compound of the present invention or its salt, andexample includes solvate of which solvent is alcohol (e.g., ethanol),hydrate, or the like. Example of hydrate includes mono-hydrate,dihydrate or the like. A solvate may be coordinated with an arbitrarynumber of solvent molecules (e.g., water molecules). The compounds orsalts thereof may be left in the atmosphere to absorb moisture, and acase where adsorbed water is attached or a case where hydrate is formedmay arise. Moreover, the compounds or salts thereof may berecrystallized to form their crystal polymorph.

As used herein, compounds that are “commercially available” may beobtained from commercial sources including but not limited to AcrosOrganics (Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis., includingSigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), AvocadoResearch (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet(Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), Crescent ChemicalCo. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman Kodak Company(Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N.H.), Maybridge Chemical Co. Ltd.(Cornwall U.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc.(Waterbury Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co.(Rockford Ill.), Riedel de Haen AG (Hannover, Germany), Spectrum QualityProduct, Inc. (New Brunswick, N.J.), TCI America (Portland Oreg.), TransWorld Chemicals, Inc. (Rockville Md.), Wako Chemicals USA, Inc.(Richmond Va.), Novabiochem and Argonaut Technology.

Compounds can also be made by methods known to one of ordinary skill inthe art. As used herein, “methods known to one of ordinary skill in theart” may be identified through various reference books and databases.Suitable reference books and treatises that detail the synthesis ofreactants useful in the preparation of compounds of the presentinvention, or provide references to articles that describe thepreparation, include for example, “Synthetic Organic Chemistry”, JohnWiley & Sons, Inc., New York; S. R. Sandler et al., “Organic FunctionalGroup Preparations,” 2nd Ed., Academic Press, New York, 1983; H. O.House, “Modern Synthetic Reactions”, 2nd Ed., W. A. Benjamin, Inc. MenloPark, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed.,John Wiley & Sons, New York, 1992; J. March, “Advanced OrganicChemistry: Reactions, Mechanisms and Structure”, 4th Ed.,Wiley-lnterscience, New York, 1992. Specific and analogous reactants mayalso be identified through the indices of known chemicals prepared bythe Chemical Abstract Service of the American Chemical Society, whichare available in most public and university libraries, as well asthrough on-line databases (the American Chemical Society, Washington,D.C., may be contacted for more details). Chemicals that are known butnot commercially available in catalogs may be prepared by customchemical synthesis houses, where many of the standard chemical supplyhouses (e.g., those listed above) provide custom synthesis services.

Although specific drugs are exemplified herein, any of a number ofalternative drugs and methods apparent to those of skill in the art uponcontemplation of this disclosure are equally applicable and suitable foruse in practicing the invention. The methods of the invention, as wellas tests to determine their efficacy in a particular patient orapplication, can be carried out in accordance with the teachings hereinusing procedures standard in the art. Thus, the practice of the presentinvention may employ conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry, and immunology within the scope of those of skill in theart. Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M.Weir & C. C. Blackwell, eds.); “Gene Transfer Vectors for MammalianCells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR: ThePolymerase Chain Reaction” (Mullis et al., eds., 1994); and “CurrentProtocols in Immunology” (J. E. Coligan et al., eds., 1991); as well asupdated or revised editions of all of the foregoing.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” thus encompass humans having pre-or early-stage inflammatory disease. Subjects may be human, but alsoinclude other mammals, particularly those mammals useful as laboratorymodels for human disease, e.g. mouse, rat, cats, dogs, horses, etc.

The expression “body fluid” as used herein in intended to include all ofthose accessible body fluids usable as clinical specimens which maycontain a compound being tested for in sufficient concentration in saidfluid to be within the limits of detection of the test device or assaybeing used. Body fluids will thus include whole blood, serum, plasma,urine, cerebrospinal fluid, synovial fluid, and interstitial and otherextracellular fluids, particularly synovial fluid of affected joints. Insome embodiments a body fluid used for determination of an abnormalmarker of early-stage inflammation is a synovial fluid from a jointsuspected of being involved in early arthritis. In other embodiments abody fluid used for marker determination is systemic, e.g. blood, urine,etc.

Care should be exercised in the collection and storage of the fluids tobe tested. Steps should be taken to avoid proteolysis of the compoundsto be tested for in said fluids, and freezing of the fluids is usuallywarranted unless the test involved can be carried out within a shortlyafter the fluids are collected. It is usually preferable to use synovialfluid rather than serum because of the likelihood that there will begreater concentrations of the compounds being tested for in the synovialfluid. On the other hand, increased levels of viscosity in synovialfluids pose problems in immunoassay systems that must be addressed bythe artisan. It may be preferable to conduct longitudinal studies of aselection of cytokines and markers as well as their respectiveinhibitors and binding proteins in order to obtain the most accurateprofile possible in determining whether an individual is in the earlystages of articular cartilage degeneration and is therefore a candidatefor intervention with the methods of the invention.

The methods of the invention can be used for prophylactic as well astherapeutic purposes. As used herein, in one embodiment the term“treating” refers to prophylactic or preventative use of theintervention in individuals with increased risk for or with early-stageinflammatory disease. In such individuals, treatment preventsdevelopment of symptoms or signs of disease, prevents development ofdisease, and/or reverses signs or symptoms of disease. In anotherembodiment, the term “treating” refers to treating individuals withestablished disease to reduce symptoms or signs of disease, to preventdisease progression, and/or to reverse symptoms of signs of disease.

Individuals at increased risk for or with early stages of aninflammatory disease are generally asymptomatic, and exhibit no orminimal symptoms and signs of the disease. In some embodiments,individuals at increased risk for developing an inflammatory disease aretreated with DHCQ to prophylactically prevent them from developing signsof an inflammatory disease, symptoms of an inflammatory disease, or theinflammatory disease. In some embodiments, individuals at increased riskfor developing an inflammatory disease are treated with DHCQ to preventthem from exhibiting progression of signs of an inflammatory disease orsymptoms of an inflammatory disease, and/or to prevent them fromdeveloping the inflammatory disease. Thus, the invention provides asignificant advance in the treatment of at-risk individuals, individualswith pre-clinical findings, or individuals with early-stage disease, bypreventing the development of clinical symptoms or signs of a disease orby preventing the progression of the clinical symptoms or signs of adisease. Such treatment is desirably performed prior to the developmentof clinical symptoms or signs of disease, and before significant loss offunction in the affected tissues, i.e. in the “at increased risk” for or“early-stage” inflammatory disease states.

In particular embodiments, the present invention provides for thetreatment of humans and other mammals that are at increased risk for,have pre-clinical, or have early-stage inflammatory disease but areasymptomatic, or have early and mild symptoms or signs of thedisease—all of these subgroups are referred to herein as patients atincreased risk of developing an inflammatory disease or a diseaseassociated with inflammation. In such asymptomatic individuals withpre-clinical or early-stage inflammatory disease, this invention canprevent the development of symptomatic inflammatory disease, prevent thedevelopment of signs of the disease, or reduce the progression ofearly-symptomatic inflammatory disease. In individuals with earlysymptoms of signs of inflammatory disease, with such early symptoms andsigns being present for less than 6 months or being mild in severity,this invention can prevent the development of the full symptoms of aninflammatory disease, prevent the development of signs and features ofthe disease, or reduce the progression of early-stage inflammatorydisease. An aspect of this invention is the treatment of asymptomaticindividuals with pre-clinical or early-stage inflammatory disease toprevent them from developing symptomatic inflammatory disease.

In various embodiments, the present invention specifically provides forthe treatment of humans and other mammals that have early-stage (whichin certain cases and diseases can have mild symptoms, or intermittentsymptoms, or symptoms for less than 6 months) orestablished-inflammatory disease. In such symptomatic individuals withearly-stage or established inflammatory disease, this invention canprevent progression of or reduce the severity of the symptoms and signsof the inflammatory disease.

In one embodiment, treatment of individuals at increased risk fordevelopment of an inflammatory disease reduced their overall risk fordevelopment of the inflammatory disease. Decreasing an individual's riskfor development of an inflammatory disease means that for an individualor a group of individuals treated with DHCQ, there will be at leastabout a 25%, at least about a 35%, at least about a 45%, at least abouta 55%, at least about a 65%, at least about a 75%, and may be around orup to about a 50% lower rate of development of the inflammatory diseaseas compared to the rate of development of the inflammatory disease inindividuals not treated with DHCQ (either previously described in theliterature for a patient population with similar characteristics, or forindividuals treated with alternative therapies).

Developing an inflammatory disease means being formally diagnosed withthe inflammatory disease by a physician. Further, developing aninflammatory disease means developing the symptoms, physical examfindings, laboratory test findings, imaging findings, and other findingsthat meet the established diagnostic criteria for the inflammatorydisease and thereby enable a physician to diagnose an individual withthe inflammatory disease.

The expression “presently or prospectively” as used herein is intendedto mean that in accordance with the methods discussed below of makingthat determination, it is possible to identify an individual as eitherbeing presently in need of such treatment, or very likely or expected tobe in need of such treatment in the near-term future. Prospective needof treatment may be established by those determinations of positivefactors that from the experience of the artisan lead directly to theearly stages of an inflammatory disease.

The expression “at increased risk of developing an inflammatory diseaseor a disease associated with inflammation” (also referred to herein as“at increased risk”) is intended to mean individuals who areasymptomatic but having an increased likelihood for developing such adisease, individuals who have mild symptoms and having an increasedlikelihood for developing such a disease, individuals who asymptomaticbut in the pre-clinical phase of such a disease, individual who havemild symptoms disease but are in the pre-clinical phase of such adisease, individuals who are asymptomatic who are in the early-stages ofdisease, and individuals who have mild symptoms who are in theearly-stages of disease. “Early stages of inflammatory disease” isintended to mean the very beginning of the initial pathologic changes.Said pathologic changes include changes in the composition, form,density, signs and/or inflammatory state and/or metabolic state of theinvolved tissue or organ as compared to that present in healthyindividuals.

Individuals at increased risk for developing an inflammatory disease ora disease associated with inflammation can be treated with DHCQ toprevent the development of disease, to prevent development of signs ofthe disease, to prevent the onset of symptomatic disease, to preventprogression of signs or symptoms of disease, or to prevent progressionof inflammation, or to prevent progression of metabolic abnormalities,or to prevent progression of associated metabolic conditions ordiseases. The DHCQ can be orally delivered using tablets, capsules or asuspension.

The retinal toxicity of HCQ significantly limits its medical use totreat humans who are at-risk for, in the pre-clinical stages of, or inthe early-stages of an inflammatory disease or disease associated withinflammation. Many physicians are reluctant to treat individualsat-risk, in the pre-clinical stages of, or in the early-stages of aninflammatory disease or disease associated with inflammation with HCQfor years or decades due to the approximately 0.5-1% risk (incidence)for the development of retinal toxicity after about 5 years oftreatment, approximately 1% risk of retinal toxicity after about 7 yearsof treatment, and approximately 2% risk of retinal toxicity after 10-15years of treatment. Individual humans who are informed by a physician orother healthcare professional that they are in the pre-clinical stagesof, or in the early-stages of, an inflammatory disease or diseaseassociated with inflammation are reluctant or unwilling to take HCQ foryears or decades to attempt to prevent development of a disease due tothe approximately 0.5-1% risk (incidence) for the development of retinaltoxicity after about 5 years of treatment, approximately 1% risk ofretinal toxicity after about 7 years of treatment, and approximately 2%risk of retinal toxicity after 10-15 years of treatment.

It is the unexpected and surprising finding that DHCQ possesses robustanti-inflammatory efficacy, while possessing minimal retinal celltoxicity, that enables DHCQ to be safely used for years or decades totreat individuals at-risk for an inflammatory disease or diseaseassociated with inflammation to prevent development of that disease. Itis the unexpected and surprising finding that DHCQ possesses robustanti-inflammatory efficacy, while possessing minimal retinal celltoxicity, that enables DHCQ to be safely used for years to decades totreat individuals in the pre-clinical stages of an inflammatory diseaseor disease associated with inflammation to prevent development of orprogression of that disease. It is the unexpected and surprising findingthat DHCQ possesses robust anti-inflammatory efficacy, while possessingminimal retinal cell toxicity, that enables DHCQ used to be safely usedfor years to decades to treat individuals in the pre-clinical stages ofan inflammatory disease or disease associated with inflammation toprevent development of or progression of that disease. With DHCQtreatment, the rates of retinal toxicity for a cumulative dose similarto that of HCQ, and over a similar dosing period, are expected to beabout 30, 40, 50, 60, or 70% lower than the rates of retinal toxicityobserved with HCQ therapy. With DHCQ therapy, the rate (incidence) ofretinal toxicity after about 5 years of treatment is expected to be lessthan 0.5%, or less than 0.25%, or less than 0.1%. With DHCQ therapy, therate of retinal toxicity after about 7 years of treatment is expected tobe less than 0.6%, or less than 0.5%, less than 0.25%, or less than0.1%. With DHCQ therapy, the rate of retinal toxicity after about 10years of treatment is expected to be less than 1%, or less than 0.75%,or less than 0.5%, less than 0.25%, or less than 0.1%. With DHCQtherapy, the rate of retinal toxicity after about 15 years of treatmentis expected to be less than 1.5%, or less than 1%, or less than 0.5%, orless than 0.25%, or less than 0.1%.

In another embodiment, this invention provides for the treatment ofindividuals with established inflammatory disease. The inflammatorydisease is diagnosed based on an individual exhibiting symptoms, signs,clinical features, laboratory test results, imaging test results, markerresults, and other findings that enable a physician to formally diagnosethat individual with the inflammatory disease. In some embodiment,established inflammatory disease is an inflammatory disease for which anindividual has had a formal diagnosis of the disease made by a physicianfor longer than 6 months. In established inflammatory disease, the signsor symptoms of disease may be more severe. In established inflammatorydisease, the disease process may cause tissue or organ damage.

Individuals at increased risk for development of an inflammatorydisease, with early-stage inflammatory disease, or with establishedinflammatory disease can be treated with DHCQ of the invention toprevent the development of disease, to prevent the progression ofdisease, and to prevent the progression of the symptoms or signs ofdisease. The dose of DHCQ is generally about 400 mg per day, or about500 mg per day, or about 550 per day, or about 600 mg per day, or about650 mg per day, or about 700 mg per day, or about 750 mg per day, orabout 800 mg per day, but can vary between 25-1600 mg per day.Inflammatory diseases and diseases associated with inflammation includeautoimmune diseases including multiple sclerosis, systemic lupuserythematosus, rheumatoid arthritis, Crohn's disease, psoriasis,autoimmune hepatitis, and other autoimmune diseases; degenerativediseases including osteoarthritis, Alzheimer's disease, maculardegeneration and other degenerative diseases; metabolic diseasesincluding type II diabetes, atherosclerosis, coronary artery disease,non-alcoholic steatohepatitis (NASH), hyperlipidemia, insulinresistance, metabolic syndrome, and other metabolic diseases; chronicinfections that result in inflammation including human immunodeficiencyvirus infection, hepatitis C virus infection, cytomegalovirus infection,and other viral, bacterial, fungal, parasite and other infection; andother inflammatory diseases such as fatty liver disease.

The present invention provides a method of treating or preventingdegeneration or destruction of articular cartilage or remodeling of thesubchondral bone in the joints of an individual in need of suchtreatment, comprising establishing the status of an individual aspresently or prospectively being in said early stages and thus in needof such treatment; and administering to the individual DHCQ, or acombination of DHCQ and atorvastatin, in an amount therapeuticallyeffective for treating or preventing said degeneration or destruction ofarticular cartilage or subchondral bone. In some embodiments thecriteria for treatment further includes evidence of inflammation in theaffected joint.

Assessment of OA may use the Kellgren Lawrence (KL) grading system(Kellgren and Lawrence, Ann. Rheum. Dis., 16:494-502, 1957, hereinspecifically incorporated by reference). The KL grading system relies onan anterior-posterior (AP) radiograph and is as follows: grade 0=nofeatures of OA; grade 1=presence of OA is doubtful, presence of minuteosteophyte(s), unchanged joint space; grade 2=minimal OA, definiteosteophyte(s), unchanged joint space; grade 3=moderate OA, moderatediminution of joint space; grade 4=severe OA, joint space greatlyreduced with sclerosis of subchondral bone. For the purposes of thepresent invention, the KL score is less than 3, in some embodiments lessthan 2, and desirably less than one.

The use of DHCQ described herein, in some embodiments, is aimed atintervention during the pre-clinical or early stages of OA, during whichthere is evidence of only mild cartilage abnormalities or lesions asdefined by the presence of at least one abnormal imaging markerindicative of pre-clinical or early-stage OA, as determined by imagingor direct visualization modalities, molecular marker analysis, orclinical history of a condition or event predisposing to the developmentof OA. The DHCQ therapy of the invention modifies OA disease progressionas measured by either stabilization of KL score and/or joint-spacenarrowing, or prevention of further cartilage breakdown (as assessed byimaging using MRI or another imaging modality), or reduction in levelsof molecular markers of cartilage breakdown.

Individuals with pre-clinical or “pre-OA” are those at increased risk ofdeveloping OA, as evidenced by abnormal inflammatory markers, abnormalbiochemical markers, abnormal imaging markers, or abnormal clinicalmarkers. Conditions or events that predispose to the development of OAinclude, without limitation, a history of injury to a joint; clinicallyor radiographically diagnosed meniscal injury with or without surgicalintervention; a ligamentous sprain with clinically or radiographicallydiagnosed anterior or posterior cruciate or medial or lateral collateralligament injury (Chu et al, Arthritis Res Ther. 2012 14(3):212. PMID:22682469); clinically measured limb-length discrepancy; obesity with acurrent, or prolonged historical period of, BMI >27; or biomechanicalfeatures of abnormal gait or joint movement. In general, a determinationof pre-clinical OA is associated with one or more, two or more, three ormore parameters (abnormal markers) of joint pathology including, withoutlimitation and relative to a healthy control sample, cartilageproteoglycan loss; cartilage damage; or elevated levels of degradativeenzymes, the presence of products of cartilage or extracellular matrixdegradation or bone remodeling. Humans at risk for OA, who have pre-OA,and who have early-stage OA are often asymptomatic, but a subset ofpatients experience joint pain due to cartilage injury (e.g. meniscalinjury), ligamentous injury (e.g. tearing of the anterior cruciateligament), or another joint abnormality. The joint pain in individualswith pre-OA and early-stage OA is generally intermittent and mild innature.

Compared to the joints of healthy control individuals, a joint in anindividual with pre-clinical OA will exhibit a KL score of 0, and haveone, two, three, four or more abnormal markers indicative ofpre-clinical disease. MRI-detected imaging markers indicative of thepresence of pre-clinical OA include cartilage edema, cartilageproteoglycan loss, cartilage matrix loss, bone marrow edema, articularcartilage fissures, articular cartilage degeneration, a meniscal tear,an anterior cruciate ligament tear, a posterior cruciate ligament tear,and other abnormalities of the cartilage or ligaments in the joint.Ultrasound will show evidence of cartilage edema or damage. Arthroscopycan allow direct detection or visualization of cartilage edema,cartilage softening, cartilage thinning, cartilage fissures, cartilageerosion, or other cartilage abnormalities. Cartilage damage isfrequently defined by the Outerbridge classification criteria or similardirectly observed changes within the joint. For example, one suchscoring system defines the presence of damage is as follows: grade0=normal cartilage; grade I: softening and swelling of cartilage; gradeII: a partial-thickness defect in the cartilage with fissures on thesurface that do not reach subchondral bone or exceed 1.5 cm in diameter;grade III: fissures in the cartilage that extend to the level ofsubchondral bone in an area with a diameter of more than 1.5 cm. Humansat risk for OA or with “pre-clinical OA” may be asymptomatic or havemild symptoms, with have a KL score of 0, but may have signs ofcartilage damage, meniscal damage, ligament damage, or otherabnormalities of the joint based on MRI imaging, ultrasound imaging, ordirect visualization of the joint on arthroscopy.

As compared to joints in healthy individuals, a joint in an individualwith early-stage OA will typically exhibit a KL score of 0 or 1 (anabnormal imaging marker), and have one, two, three, four or moreabnormal markers indicative of early disease. Plain X-rays of theinvolved joint would demonstrate features consistent with a KL score of0-2, including no osteophytes or small osteophytes, and no or minimaljoint space narrowing. MRI-detected imaging markers indicative ofearly-stage OA include cartilage proteoglycan loss, cartilage thinning,cartilage fissures or cartilage breakdown. Ultrasound will show evidenceof cartilage edema or damage. Arthroscopy can provide for directdetection or visualization of cartilage edema, cartilage softening,cartilage thinning, cartilage fissures, cartilage erosion, or othercartilage abnormalities. Cartilage damage is frequently defined by theOuterbridge classification criteria or similar direct observationalchanges within the joint. Humans with early OA may be asymptomatic, ormay have mild or intermittent symptoms, or may have symptoms for lessthan 6 months, but may exhibit findings associated with cartilage damageas represented by Outerbridge grade 0, grade I and grade II scores orsimilar direct observational changes within the joint, as well as withother cartilage, meniscal and ligament damage based on MRI imaging,ultrasound imaging, or direct visualization of the joint on arthroscopy.

In contrast to pre-clinical (also termed at-risk for) OA and early-stageOA, established or advanced OA can be defined radiographically as KLgrade >=2 or as MRI evidence of extensive, complete, or near-completeloss of articular cartilage. Other marker evidence of joint failure canbe determined by direct or arthroscopic visualization of extensive,complete, or near-complete loss of joint space or cartilage, bybiomechanical assessment of inability to maintain functional jointintegrity, or by clinical assessment of joint failure, as evidenced byinability to perform full range of motion or to maintain normal jointfunction. On physical examination, patients with advanced OA can haveabnormal clinical markers including bony enlargement, small effusions,crepitus, and malalignment of the synovial joints. Examples ofsemiquantitative MRI scoring systems that can be used to classify theseverity of OA include: WORMS (Whole-Organ Magnetic Resonance ImagingScore; Peterfy C G, et al. Osteoarthritis Cartilage 2004; 12:177-190);KOSS (Knee Osteoarthritis Scoring System; Kornaat P R, et al. SkeletalRadiol 2005; 34:95-102); BLOKS (Boston Leeds Osteoarthritis Knee Score;Hunter D J, et al. Ann Rheum Dis 2008; 67:206-211); MOAKS (MRIOsteoarthritis Knee Score; Hunter D J, et al. Osteoarthritis Cartilage.2011; 19(8):990-1002); HOAMS (Hip Osteoarthritis MRI Score; Roemer F W,et al. Osteoarthritis Cartilage. 2011; 19(8):946-62); OHOA (Oslo HandOsteoarthritis MRI Score). Advanced OA can result in significant jointpain and loss of mobility owing to joint dysfunction.

In a preferred embodiment, the individual treated with the compositionsand by the methods of the invention has pre-clinical (at-risk) orearly-stage OA or RA. In other embodiments, the individual treated bythe methods of the invention has established OA, RA or other type ofarthritis.

A variety of markers can be used to assess inflammation in pre-clinicalOA, early-stage OA, and advanced OA, including imaging markers,molecular markers, and clinical markers. Examples of abnormal clinicalmarkers include the presence of a joint effusion on physicalexamination. Another example of an abnormal clinical marker is thepresence of morning stiffness in the joint. Examples of abnormal imagingmarkers include the use of MRI or ultrasound-detected signs ofinflammation in the joint. MRI can be performed either with or withoutgadolinium contrast, and MRI-evidenced inflammation is defined as thepresence of one or more of the following findings: synovitis (synoviallining thickening, proliferation, and/or enhancement (increased signal),including a positive Doppler-flow signal in the synovial lining), jointeffusion, bone marrow edema, etc (Krasnokutsky et al, Arthritis Rheum.2011 63(10):2983-91. doi: 10.1002/art.30471 PMID: 21647860; Roemer etal, Osteoarthritis Cartilage. 2010 October; 18(10):1269-74. PMID:20691796; Guermazi et al, Ann Rheum Dis. 2011 70(5):805-11, PMID:21187293). Ultrasound-evidenced inflammation (an abnormal imagingmarker) is defined as the presence of one or more of the followingfindings: synovial lining thickening and/or enhancement, a jointeffusion, bone marrow enhancement, etc. (Guermazi et al, Curr OpinRheumatol. 2011 23(5):484-91. PMID: 21760511; Hayashi et al,Osteoarthritis Cartilage. 2012 March; 20(3):207-14. PMID: 22266236;Haugen et al, Arthritis Res Ther. 2011; 13(6):248. PMID: 22189142).Molecular markers that can be used to assess inflammation includeerythrocyte sedimentation rate (ESR), CRP, cytokines, chemokines, andother inflammatory mediators. ESR and CRP are measured in blood, and theother molecular markers of inflammation can be measured in blood orsynovial fluid.

In one embodiment, one or more of these inflammatory markers includingabnormal physical exam markers, abnormal imaging (MRI findings,ultrasound findings) markers, abnormal laboratory markers (CRP, ESR),and other abnormal biomarkers are used to identify individuals withactive inflammation that are most likely to respond to treatment withDHCQ, or DHCQ+atorvastatin. In another embodiment, individuals withdegenerative meniscal tear of the knee are subjected to MRI analysis ofthe knee and hs-CRP laboratory testing. If the MRI synovitis score(Guermazi et al., Ann Rheum Dis. 2011 70(5):805-11. PMID: 21187293) ismeasured to be >5 or the hs-CRP is measured to be >2.5 mg/L, then theindividual is determined to be at increased risk for or havingearly-stage OA and is thus treated with DHCQ. In another embodiment,individuals at increased risk for knee OA who experience intermittentknee pain are subjected to MRI analysis of the knee and hs-CRPlaboratory testing. If the MRI synovitis score (Guermazi et al., AnnRheum Dis. 2011 70(5):805-11. PMID: 21187293) is measured to be >5 orthe hs-CRP is measured to be >2.5 mg/L, then the individual isdetermined to be at increased risk for or having the early-stages of OAand is therefore treated with DHCQ.

In another embodiment, one or more of these same inflammatory markers isused to monitor an individual's response to treatment, to determine iftreatment should be continued, or to determine if treatment can bediscontinued. For example, individuals at increased risk for OA who arebeing treated with DHCQ are monitored annually, or every-other year, byMRI and hs-CRP. Individuals, whose MRI synovitis score declines to below3 or whose hs-CRP declines to below 1 mg/L are identified as havingexhibited a positive response to therapy and that their at-risk state,early-disease state, or established disease state has responded well totreatment.

Individuals at increased risk for the development of RA, or with“pre-clinical RA”, or with early-stage RA, are identified based on thepresence of abnormal inflammatory, abnormal imaging, or abnormalclinical markers indicative of RA. Abnormal markers that suggest anindividual has early-stage RA include one or more of the following:presence of one or more swollen joints, presence of anti-CCP or RFantibodies, evidence of synovial enhancement (increased signal) on MRIscan or ultrasound, elevated levels of autoantibodies or cytokines thathave can predict the development of RA (as described in Sokolove et al,PLoS One. 2012; 7(5):e35296; Deane et al, Arthritis Rheum. 201062(11):3161-72; Gerlag et al, Ann Rheum Dis. 2012 71(5):638-41). Factorsthat increase an individual's risk of developing RA include one or moreof the following: a family history of RA (particularly in a first-degreerelative), increased levels of anti-CCP and/or RF autoantibodies, agenetic profile associated with susceptibility to RA, and cigarettesmoking (as described in Deane et al, Rheum Dis Clin North Am. 201036(2):213-41; Klareskog et al, Semin Immunol. 2011 April; 23(2):92-8).

Individuals are classed as being at risk of developing RA on the basisof their being measured to have specific abnormal biochemical,serologic, genetic, imaging, or clinical markers. The pre-clinical phaseof RA is characterized by the presence of abnormal inflammatory markersof RA, including the development of anti-citrullinated proteinantibodies (ACPA) and rheumatoid factor (RF) years before the onset ofclinically apparent RA. As the onset of clinical apparent diseaseapproaches, the ACPA response spreads, i.e., there is an increase innumber of levels of autoantibodies targeting citrullinated proteins.Additionally, there is often a concomitant rise in the level of serumcytokines and chemokines as well as acute phase reactants (including butnot limited to ESR and CRP) (Sokolove et al, PLoS One. 012; 7(5):e35296.2012, PMID: 22662108; Deane et al, Arthritis Rheum. 2010 November;62(11):3161-72). Thus, “at risk” and “pre-clinical” RA can be defined bythe presence of the molecular markers ACPA, RF, elevated cytokines, orcombinations of these markers. Additionally, pre-clinical RA including“at risk” could be defined by genetic markers and/or family history.Such genetic markers are considered abnormal markers herein and includebut are not limited to the HLA DR4 shared epitope and other geneticpolymorphisms, such as PTPN22, PAD4, STAT4, and TRAF1-C5.

Early-stage RA is rarely asymptomatic; it most often manifests as painin and/or stiffness of the small or medium joints, and it can beassociated with joint swelling or synovitis. Early-stage RA can bedefined by the presence of signs and symptoms and abnormal markersconsistent with RA of less than 3-6 months duration and lack ofradiographic joint damage as determined by plain X-ray. Early-stage RAis also indicated by the presence of abnormal imaging markers(determined, for example, by MRI or ultrasound, including increasedDoppler-flow signal on ultrasound), such as synovial enhancement, bonemarrow edema, an effusion, or other findings indicative of inflammation(Gerlag et al, Ann Rheum Dis. 2012 71(5):638-41. PMID: 22387728).

Advanced RA is can be defined as RA of greater than 3-6 months durationand often at last 1 year duration. Radiographic signs of RA (abnormalimaging markers), such as periarticular erosions of the bone, can bedetected within 1-2 years of disease onset, and therefore an alternativedefinition of advanced RA may include evidence of radiographicjoint-space narrowing and/or erosions.

Multiple sclerosis is an autoimmune neurologic condition caused bydemyelination of neurons as a result of immune injury. It is caused by adirect immunologic attack, mediated by autoreactive T cells and B cells,on protein and lipid components of the myelin sheath.

Individuals with pre-clinical MS are those at increased risk ofdeveloping MS, as indicated by abnormal biochemical, serologic, genetic,imaging, or clinical parameters. The pre-clinical phase of MS can becharacterized by the presence of abnormal inflammatory markersassociated with the later onset of MS, for example autoantibodies thatappear several years before the onset of clinically apparent MS.Additionally or alternatively, individuals with pre-clinical MS can haveneurologic signs and/or symptoms that alone do not diagnose MS but maybe associated with the later onset of clinically apparent MS. Such signsor symptoms include but are not limited to optic neuritis (whichgenerally manifests as loss of vision or decreased vision in one eye),numbness, dizziness, muscle spasms. Symptoms of pre-clinical MS aretypically of limited duration but can wax and wane. They may beassociated with radiographic changes including but not limited towhite-matter lesions as determined by MRI, which often appear as brightareas on T2-weighted MRI. Additionally, pre-clinical MS can beassociated with the presence of cerebrospinal fluid (CSF) abnormalitiesincluding abnormally high numbers of white blood cells or levels ofprotein, and/or the presence of oligoclonal bands.

Thus, pre-clinical MS can be defined as the presence of clinicalsymptoms of early demyelination and/or by the presence of specificautoantibodies in serum or CSF, abnormally high levels of protein orwhite blood cells in CSF, brain or spinal cord lesions detected byimaging, or combinations of these markers.

Early-stage MS most often manifests as persistent or recurrentneurologic symptoms of demyelination, including but not limited to focalor multifocal numbness, tingling, weakness, loss of balance, orcompromised vision including blurry or double vision. Definitivediagnosis of MS requires evidence of 2 or more brain lesions detected byMRI and/or 2 or more episodes of neurologic symptoms lasting at least 24hours and occurring at least one month apart.

Advanced MS can be defined as MS that has progressed to permanentneurologic disability, usually with non-resolving lesions as detected byMRI. Additionally, MS symptoms may wax and wane in a pattern known asrelapsing-remitting MS. This pattern can be seen late into the course ofMS, with or without continued accrual of damage in a chronic progressivepattern in which disease progresses with increasing neurologic symptomswithout complete recovery from prior lesions.

Atherosclerosis is characterized by accumulation of fatty materials inthe arterial wall, resulting in development of fatty plaques, which mayrupture and cause vascular occlusion and ischemia. The lesion ofatherosclerosis comprises a highly inflammatory milieu characterized bythe accumulation of inflammatory cells, including macrophages and to alesser extent T and B cells, and production of high levels ofinflammatory cytokines, chemokines, and MMPs (Libby et al, Nature 2011.473(7347):3170-25. PMID#21593864). Atherosclerosis is associated withand likely promoted by low-grade inflammation.

Individuals at risk for the development of atherosclerosis are thosewith known risk factors for atherosclerotic coronary artery disease.Risk factors include traditional risk factors for atherosclerotic heartdisease, such as those described in the Framingham Risk Score, andincluding the following abnormal markers: high blood pressure, cigarettesmoking, elevated levels of HDL cholesterol, glucose intolerance,increased age, male sex, and other factors (see D'Agostino R B Sr, VasanR S, Pencina M J, Wolf P A, Cobain M, Massaro J M, Kannel W B.Circulation. 2008 Feb. 12; 117(6):743-53. PMID: 18212285).

Early-stage atherosclerosis is characterized by early changes incoronary arteries, cerebral arteries, and/or other arteries. Sucharterial abnormalities can be visualized through imaging using MRI, CT,angiography, or other methods. Because such early-stage disease does notocclude the involved blood vessels, individuals are asymptomatic andthey exhibit normal exercise (treadmill or bicycle) or chemical(persanthine or adenosine or dobutamine) stress test results (based onreadouts using radiographic contrast and/or electrocardiogram (EKG)changes suggestive of ischemia).

Advanced atherosclerosis is characterized by symptomatic heart orcardiovascular disease, including angina, myocardial infarction,transient ischemic attacks, and/or stroke due to arterial occlusion.Advanced atherosclerosis manifests as more advanced arterialabnormalities that can be visualized as abnormal imaging markers throughimaging using MRI, CT, angiography, and other methods. In addition, withadvanced atherosclerosis functional testing with an exercise (treadmillor bicycle) or chemical (persanthine or adenosine or dobutamine) stresstest results findings suggestive of ischemia detected by radiographiccontrast and/or electrocardiogram (EKG).

In one embodiment, one or more abnormal markers of inflammation are usedto identify individuals at increased risk for atherosclerotic diseaseand exhibiting active inflammation, and thus likely to respond totreatment with DHCQ, or a combination of DHCQ+atorvastatin. In anotherembodiment, individuals at increased risk for atherosclerotic diseasewith increased blood cholesterol (total cholesterol >250 mg/dL orLDL >150 mg/dL) are subjected to hs-CRP laboratory testing. If thehs-CRP is >3, then the individual is determined to be at high-risk forprogression of atherosclerotic heart disease and is treated with DHCQ,or the combination of DHCQ and atorvastatin.

In another embodiment, hs-CRP is used to monitor an individual'sresponse to treatment with DHCQ, or the combination of DHCQ andatorvastatin, to determine if the individual who is at increased riskfor atherosclerotic disease has responded to treatment and/or if thetreatment should be continued. For example, individuals at increasedrisk for atherosclerotic who are being treated with DHCQ, or thecombination of DHCQ and atorvastatin, are monitored annually, orevery-other year, by repeat cholesterol and hs-CRP testing. Individuals,whose total cholesterol declines below 220, LDL cholesterol declines tobelow 120, and whose hs-CRP declines to be below 1 are identified ashaving exhibited a positive response to therapy and that their at-riskstate, early-disease state, or established disease state iswell-controlled by DHCQ, or combination therapy with DHCQ+atorvastatin.

Type II diabetes mellitus is characterized by the presence of insulinresistance and hyperglycemia, which can cause retinopathy, nephropathy,neuropathy, or other morbidities. Insulin resistance (IR), which isreferred to as a disease state herein, is a physiological condition inwhich cells fail to respond to the normal actions of the hormoneinsulin. The body produces insulin, but the cells in the body becomeresistant to insulin and are unable to use it as effectively, leading tohyperglycemia. Beta cells in the pancreas subsequently increase theirproduction of insulin, further contributing to hyperinsulinemia. Thisoften remains undetected and can contribute to a diagnosis of Type 2Diabetes. Additionally, type 2 diabetes is a well-known risk factor foratherosclerotic cardiovascular disease. Metabolic syndrome refers to agroup of factors, including hypertension, obesity, hyperlipidemia, andinsulin resistance (manifesting as frank diabetes or high fasting bloodglucose or impaired glucose tolerance), that raises the risk ofdeveloping heart disease, diabetes, or other health problems; (Grundy etal, Circulation. 2004; 109:433-438). There is a well-characterizedprogression from normal metabolic status to a state of “insulinresistance” as assess by impaired fasting glucose (IFG: fasting glucoselevels greater than 100 mg/dL) or to a state of impaired glucosetolerance (IGT: two-hour glucose levels of 140 to 199 mg/dL after a 75gram oral glucose challenge). Both IFG and IGT are considered abnormalmetabolic markers indicative of a pre-diabetic states, with over 50% ofsubjects with IFG progressing to frank type II diabetes within, onaverage, three years (Nichols, Diabetes Care 2007. (2): 228-233). Theinsulin resistance is caused, at least in part, by chronic low-gradeinflammation (Romeo G R et al, Arterioscler Thromb Vasc Biol. 201232(8):1771-6; de Luca C et al, FEBS Lett. 2008 582(1):97-105; Ma K etal, Diabetes Metab Res Rev. 2012 28(5):388-94).

Pre-clinical type II diabetes or “at risk” for type II diabetes can bedefined as impaired fasting glucose, which is defined as a fastingglucose greater than 100 mg/dL. Humans with impaired fasting glucoselevels and who are “at risk” of developing type II diabetes areasymptomatic.

Early-stage type II diabetes is defined by an abnormal fasting bloodglucose reading of >126 mg/dL on two separate occasions. Individualswith early-stage type II diabetes do not have symptoms or signs oftissue damage or end-organ damage.

Advanced type II diabetes is characterized by abnormal metabolic markersincluding persistent elevation in blood glucose levels over 200 mg/dL ina non-fasting state, or multiple readings of >126 mg/dL in the fastingstate, and a hemoglobin A1c reading of >7%. Humans with advanced type IIdiabetes frequently have symptoms, microvascular complications, and/orend-organ or tissue damage.

In one embodiment, measurement of one or more abnormal metabolic andinflammatory markers are used to identify individuals at increased riskfor type II diabetes whom have active inflammation, and thus are athighest risk for progression of their type II diabetes and also mostlikely to respond to treatment with DHCQ, or the combination ofDHCQ+atorvastatin. For example, individuals with a body mass index (BMI)greater than 25 are tested for their fasting blood glucose, hemoglobinA1c, and hs-CRP. Individuals who exhibit a fasting blood glucose >126mg/dL on two separate occasions, and who have either a hemoglobinA1c >6.5% or a hs-CRP >3 mg/L, are identified as exhibiting abnormalmetabolic marker(s) and are at highest risk for progression of diseaseand initiated on therapy with DHCQ, or the combination of DHCQ andatorvastatin.

In another embodiment, one or more of these same metabolic andinflammatory markers are used to monitor an individual's response totreatment, to determine if treatment needs to be continued, or todetermine if treatment can be discontinued. For example, individuals atincreased risk for type II diabetes who are being treated with DHCQ, orthe combination of DHCQ and atorvastatin, are monitored annually, bytesting for hemoglobin A1c and hs-CRP. Individuals, whose hemoglobin A1cdeclines to less than 5.6% and hs-CRP declines to below 1 are identifiedas having exhibited a positive response to therapy and that theirat-risk state, early-disease state, or established disease state hasresponded well to treatment.

The International Diabetes Federation consensus worldwide definition ofthe term metabolic syndrome (2006) is based on the followingabnormalities in inflammatory markers, metabolic markers and clinicalmarkers: Central obesity (defined as waist circumference# withethnicity-specific values) AND any two of the following: Raisedtriglycerides: >150 mg/dL (1.7 mmol/L), or specific treatment for thislipid abnormality; Reduced HDL cholesterol: <40 mg/dL (1.03 mmol/L) inmales, <50 mg/dL (1.29 mmol/L) in females, or specific treatment forthis lipid abnormality; Raised blood pressure (BP): systolic BP >130 ordiastolic BP >85 mm Hg, or treatment of previously diagnosedhypertension; Raised fasting plasma glucose (FPG): >100 mg/dL (5.6mmol/L), or previously diagnosed type 2 diabetes. The World HealthOrganization 1999 criteria require the presence of any one of diabetesmellitus, impaired glucose tolerance, impaired fasting glucose orinsulin resistance, AND two of the following: Blood pressure: ≥140/90mmHg; Dyslipidemia: triglycerides (TG): ≥1.695 mmol/L and high-densitylipoprotein cholesterol (HDL-C)≤0.9 mmol/L (male), ≤1.0 mmol/L (female);Central obesity: waist:hip ratio >0.90 (male); >0.85 (female), or bodymass index >30 kg/m2; and Microalbuminuria: urinary albumin excretionratio ≥20 μg/min or albumin:creatinine ratio ≥30 mg/g. Associateddiseases and signs are: hyperuricemia, fatty liver (especially inconcurrent obesity) progressing to NAFLD, polycystic ovarian syndrome(in women), and acanthosis nigricans. Progression of metabolic syndromeresults in frank diabetes or high fasting blood glucose or impairedglucose tolerance, and as a result individuals develop the symptoms andsigns of coronary artery disease, type II diabetes, heart disease,diabetes, or other health problems. In one embodiment, DHCQ is used totreat patients with the metabolic syndrome with resultant reduction inserum lipids (inducing reduced total cholesterol, LDL, or triglyceridesor alternatively, increased HDL). In another embodiment, DHCQ is used totreat patients with the metabolic syndrome with resultant reduction ininsulin resistance as manifest by reduced levels of fasting serumglucose, reduced levels of post-prandial serum glucose, and/or reductionin hemoglobin A1c. In another embodiment, DHCQ is used to treat patientswith hyperlipidemia with resultant reduction in total cholesterol, orLDL, or triglycerides or alternatively, increased HDL.

Metabolic syndrome is an inflammatory condition. In the setting of themetabolic syndrome, macrophages accumulate in obese adipose tissue,where they produce TNF and other inflammatory cytokines in response tostimulation with saturated fatty acids and circulatinglipopolysaccharide (LPS) (Johnson et al, Cell 2013. 152(4):673-84;Bhargava P et al, Biochem J. 2012 442(2):253-62). Moreover, TNFinhibition can abrogate insulin resistance (Johnson et al, Cell 2013.152(4):673-84), however this is not practical or safe for long termtherapy. Thus, the metabolic syndrome is a metabolic disease which isdirectly or indirectly associated with co-morbid inflammation. Asdiscussed above, most commonly used anti-inflammatory therapeuticstherapies themselves possess potential hepatoxicity (i.e.corticosteroids, methotrexate, leflunomide, sulfasalazine) or thepotential to worsen the components of the metabolic syndrome (i.e.calcineurin inhibitors such as tacrolimus and cyclosporine areassociated with hypertension, hyperlipidemia, and diabetes as well assmall potential risk of hepatotoxicity).

In one embodiment, one or more metabolic and inflammatory markers areused to identify individuals at increased risk for metabolic syndromeand who have active underlying disease, and thus are at high risk fordisease progression and most likely to respond to treatment with DHCQ,or the combination of DHCQ+atorvastatin. For example, individuals with abody mass index (BMI) greater than 25 are tested for two or more of thefollowing metabolic markers: fasting blood glucose, hemoglobin A1c,total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, andblood pressure. Individuals who have a fasting blood glucose >126 on twooccasions, and at least 1 of the following signs or findings, or atleast 2 of the following signs or findings, or at least 3 of thefollowing signs or findings, are identified as having abnormal metabolicmarker(s) and thus being at high-risk for development of metabolicsyndrome and are initiated on treatment with DHCQ, or the combination ofDHCQ+atorvastatin. Signs or findings include but are not limited to thefollowing abnormal inflammatory markers, metabolic markers, and clinicalmarkers: Blood pressure: ≥140/90 mmHg; Triglycerides (TG): ≥1.695 mmol/Land high-density lipoprotein cholesterol (HDL-C)≤0.9 mmol/L (male), ≤1.0mmol/L (female); or Microalbuminuria: urinary albumin excretion ratio≥20 μg/min or albumin:creatinine ratio ≥30 mg/g.

In another embodiment, one or more of these same metabolic andinflammatory markers are used to monitor an individual's response toDHCQ treatment or to DHCQ+atorvastatin treatment, to determine iftreatment should be continued, or to determine if treatment can bediscontinued. For example, individuals at increased risk for metabolicsyndrome who are being treated with DHCQ, or the combination of DHCQ andatorvastatin, are monitored annually. Individuals, who exhibit one ormore of the following are identified as having exhibited a positiveresponse to therapy: fasting blood glucose returns to less than 126mg/dL, hemoglobin A1c declines to less than 5.6%, blood pressure becomesless than 140/90 mmHg, triglycerides (TG) below 1.695 mmol/L,high-density lipoprotein cholesterol (HDL-C) increases, andmicroalbuminuria:urinary albumin excretion ratio normalizes. Individualswho exhibit a positive response to therapy based on improvement in oneor more signs and findings are identified as having exhibited a positiveresponse to therapy, and that their at-risk state, early-disease state,or established metabolic disease state has responded well to treatmentwith DHCQ, or to treatment with DHCQ+atorvastatin.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholicsteatohepatitis (NASH) are conditions associated with fatty infiltrationof (or accumulation in) the liver. Although fatty infiltration alonedoes not cause liver damage, when it is accompanied by an inflammatoryreaction it can lead to fibrosis and liver cirrhosis and ultimatelyhepatic failure. The inflammation in NASH is characterized byinfiltration of the liver by macrophages and lymphocytes, as well asalterations in the liver's macrophage-like Kupfer cell population (Tilg,et al, 2010. Hepatology. 52(5):1836-46). Inflammatory cytokines,particularly TNF, are central to the pathology of NASH. The source ofTNF is unclear: it may be peripheral, i.e., inflammatory adipose tissue,or local, i.e., innate immune cells activated by portal-derivedendotoxin or by free fatty acid (Tilg et al, 2010. Hepatology.52(5):1836-46). The endotoxin-responsive TLR4 receptor has been shown tobe critical to disease in a mouse model of NASH (Tsukumo et al, Diabetes2007. 56(8):1986-98).

There are no currently approved drug treatments for NASH. Dietmodification, weight loss, and gastric bypass resulting in the priorhave shown the most efficacy in treatment of NASH. Small to moderatesized studies of metformin, pioglitazone, and vitamin E have shown smallbut significant benefits. However, results have not been consistent withmany studies ultimately failing to show an ultimate change in fibrosis(Schwenger K J P, World J Gastroenterol. Feb. 21, 2014; 20(7):1712-1723.). Given that inflammation is a major component of NASH, itwould be anticipated that anti-inflammatory or immunosuppressivetherapies could offer efficacy in the treatment of NASH. However, mostcommonly used anti-inflammatory and immunosupressive therapiesthemselves possess potential hepatoxicity. For instance, corticosteroidsare associated with development of hepatic steatosis and non-steroidalanti-inflammatory agents are associated with cholestasis and arecontraindicated in the setting of cirrhosis. Similarly other drugscommonly used in systemic inflammatory diseases including methotrexate,leflunomide, and sulfasalazine are not uncommonly associated withhepatotoxicity. Likewise, other commonly used immunosuppressants areassociated with many of the common co-morbidities seen in patients withNASH. For example, calcineurin inhibitors such as tacrolimus andcyclosporine are associated with hypertension, hyperlipidemia, anddiabetes as well as small potential risk of hepatotoxicity.

Pre-clinical NASH or “at risk” for NASH can be defined as NAFLD, whichis the presence of fatty infiltration of the liver on ultrasound (anabnormal imaging marker) in the absence of alcohol consumption orexposure to other liver toxins. The abnormal imaging marker of fattyinfiltration of the liver is assessed by ultrasound imaging of theliver, and determination that an individual patient exhibits ultrasoundresults consistent with fatty infiltration of the liver which areoutside of the range of ultrasound findings in 95% of normal humans.Humans with NAFLD and who have pre-clinical NASH (i.e., NAFLD) havenormal levels of liver enzymes in their blood (e.g. normalaminotransferase [transaminase] levels, including a normal AST (SGOT)and ALT (SGPT)).

Early-stage NASH is defined as the presence of NAFLD in conjunction withhepatic inflammation and injury, as reflected by abnormally high levelsof blood aminotransferases (i.e., elevated levels of AST (SGOT) and ALT(SGPT) as compared to the normal range in humans—which representabnormal markers of inflammation for NASH).

Advanced NASH is defined as the presence of chronic liver inflammationand injury, as reflected by persistently elevated levels of livertransaminases (persistently elevated AST (SGOT) and ALT (SGPT)), and thepresence of early or advanced hepatic fibrosis and/or cirrhosis. Hepaticfibrosis is identified by ultrasound or CT or MRI imaging of the liver,or by liver biopsy.

In one embodiment, measurement of one or more abnormal metabolicmarkers, abnormal inflammatory markers, and abnormal imaging markers areused to identify individuals at increased risk for subsequentdevelopment of NAFLD or NASH, and thus are most likely to respond totreatment with DHCQ, or the combination of DHCQ and atorvastatin. Forexample, individuals with elevated liver transaminases, based on AST >60IL/L (normal range 6-40 IU/L) or ALT >50 IU/L (normal range 7-35 IU/L),ultrasound findings indicative of fatty liver, and a fasting bloodglucose >126 on two separate readings, or hemoglobin A1c >6.5%, areidentified as exhibiting abnormal inflammatory markers, abnormal imagingmarkers or abnormal metabolic markers and therefore being at high riskfor progression to NASH and initiated on therapy with DHCQ, or thecombination of DHCQ and atorvastatin.

In another embodiment, one or more of these same metabolic andinflammatory markers are used to monitor an individual's response totreatment, to determine if the individual has exhibited a positiveresponse to therapy, or to determine if treatment can be discontinued.For example, individuals at increased risk for NAFLD or NASH who arebeing treated with DHCQ, or the combination of DHCQ and atorvastatin,are monitored periodically, by testing for AST, ALT, hemoglobin A1c andfasting blood glucose. Individuals, whose AST and ALT normalize, whosehemoglobin A1c declines to less than 5.6%, and whose fasting bloodglucose normalizes are identified as having exhibited a positiveresponse to therapy, and that their at-risk state, early-disease state,or established disease state has responded well to treatment.

Given the co-occurrence of hyperlipidemia and insulin resistance in themetabolic syndrome, DHCQ could provide a unique and unexpected role asan anti-inflammatory and anti-metabolic which would be well tolerated inthe demographic of patients affected by or at risk for development ofmetabolic syndrome. A potential risk to use of HCQ for treatment ofmetabolic syndrome would be potential retinal toxicity associated withuse of HCQ in the setting of type 2 diabetes. Current recommendationsare that HCQ be used with caution or avoided in those with increasedrisk for retinal toxicity, including those with underlying retinaldisease, those with diabetes, the overweight, or those over age 60.Thus, the identification of an agent in possession of theimmunomodulatory and metabolic properties of HCQ but with reduced riskof retinal toxicity could provide a viable therapeutic for the metabolicsyndrome, including both its metabolic and inflammatory components. Anexample would be treatment of a patient with metabolic syndrome withDHCQ which would potentially allow not only adequate initial dosing oftherapy but could additionally allow dose titration to a level whichachieves clinical efficacy, including dosing at level higher than therecommended dose limitations for HCQ. Anticipated results would besuppression of systemic inflammation with concomitant reductions inserum lipids and improvement in insulin sensitivity as well as reductionor normalization of hepatic enzymes in the setting of concurrent NASH.

The disclosed compositions and methods involving DHCQ can also be usedto treat alcoholic steatohepatitis. Prolonged consumption of significantamounts of alcohol can result in a range of symptoms from simplesteatosis to cirrhotic liver failure. In fact, nearly 90% of individualswho consume more than 60 grams of alcohol a day develop will developfatty infiltration of the liver. This steatosis can progress to chronicalcoholic hepatitis which can range from mild and chronicallyprogressive disease to a severe and fulminant necrohepatitis. Though themechanisms of alcoholic hepatitis are likely multifactorial, there is arole for inflammation involving multiple pathways including the innateimmune receptors as well as cytokines such as TNFα and IL-1β. Acutealcoholic hepatitis has been treated with a range of anti-inflammatorytherapies including corticosteroids as well as TNFα blockers (O'Shea etal, Alcoholic Liver Disease, Hepatology, 2010, 51(1):307-28). Thoughpotentially effective in the acute setting of severe alcoholichepatitis, the risk-benefit profile of such therapies prohibits use forchronic alcoholic steatohepatitis. Given the critical role forinflammation including innate immune activation via TLRs, the disclosedcompositions and methods involving DHCQ can also be used to treatchronic alcoholic steatohepatitis. This is particularly critical ashepatic insufficiency is a risk factor for retinal toxicity withhydroxychloroquine and thus a drug with a wider therapeutic window withrespect to retinal toxicity would be preferred.

The following provides examples of approaches to determining whetherinflammation is present in an individual, including individuals at riskfor a variety of different inflammatory diseases, such as autoimmunediseases (e.g., RA, MS, Crohn's disease, psoriasis, etc), degenerativediseases involving low-grade inflammation (e.g., OA, Alzheimer'sdisease, macular degeneration, etc), other inflammatory diseases (e.g.,NASH, type II diabetes, metabolic syndrome, atherosclerosis, cardiacdisease, etc.), as well as inflammatory diseases associated with chronicinflammation (e.g., HIV infection, HCV infection, CMV infection, TBinfection, etc.). Although the following describes the approach toidentifying inflammation particularly in humans at risk of developingarthritis or with early-stage arthritis, in another embodiment it is useto assess disease activity or tissue or organ damage in individuals withestablished inflammatory disease.

A variety of markers can be used to assess inflammation in inflammatorydiseases, including imaging markers, molecular markers, and clinicalmarkers. Measurement or detection of abnormal levels or values orfindings of such markers can facilitate identification of individuals atincreased risk of developing or in an early-stage of an inflammatorydisease or disease associated with inflammation, and can be used toidentify individuals who would benefit from treatment with DHCQ, orDHCQ+atorvastatin.

Many inflammatory diseases and diseases associated with inflammation,including those described above, are associated with metabolicabnormalities, as reflected by abnormal metabolic marker levels orvalues. Most anti-inflammatory/immunosuppressive therapies which wouldbe used to treat such disorders also lack significant capability toameliorate the metabolic abnormalities observed with inflammatorydisease and many, including corticosteroids, and calcineurin inhibitorscan themselves induce metabolic abnormalities including insulinresistance, hypertension, and hyperlipidemia. Similarly, given theincreased risk of underlying liver disease due to NAFLD/NASH among thosewith metabolic syndromes, immunosuppressives such as methotrexate andleflunomide should be avoided due to their potential intrinsichepatotoxicity. Similarly, HCQ, which may have a favorable metabolicprofile is itself associated with risk of retinal toxicity and thusshould be used cautiously or avoided in those at increased risk forunderlying retinal disease including those with type 2 diabetes. Thususe of DHCQ has a unique ability to treat not only inflammation but alsothe metabolic components of inflammatory disease including but notlimited to insulin resistance, hyperlipidemia, and NASH. And DHCQ can doso with a reduced risk of retinal toxicity. Thus, in one embodiment,DHCQ could be used to treat inflammation with associated metabolicdisease including but not limited to insulin resistance, type 2diabetes, hyperlipidemia, and NAFLD and/or NASH. In another embodiment,DHCQ could be used to treat inflammation with associated metabolicdisease which placed the patient at increased risk for retinal toxicity.Such properties of DHCQ are unexpected in that both reduced retinaltoxicity and favorable metabolic effects are lacked by the othermetabolites of the parent molecule HCQ.

A comprehensive description of the markers (including inflammatorymarkers, metabolic markers, clinical markers and imaging markers) for OAand RA are presented as an example of how one approaches developingmarkers for a pre-clinical or early-stage inflammatory disease ingeneral. In arthritis, examples of abnormal clinical markers includewarmth, erythema (redness), inflammation, and effusions. Other examplesof abnormal clinical markers are morning stiffness in the joint lastingmore than 1 hour, and pain and swelling. Examples of abnormal imagingmarkers include MRI- or ultrasound-detected inflammation in the joint.MRI, performed with or without gadolinium contrast, detects inflammation(an abnormal imaging marker) on the basis of the presence of one or moreof the following findings: synovitis (synovial lining thickening,proliferation and/or enhancement (increased signal on Gd-MRI));increased Doppler-flow signal in the synovial lining); a joint effusion;extensive bone marrow edema; and other findings suggestive ofinflammation. When ultrasound is the imaging method used, inflammation(an abnormal imaging marker) is defined by the presence of one or moreof the following findings: synovial lining thickening and/orenhancement, a joint effusion, bone marrow enhancement, and otherfindings suggestive of inflammation. Molecular markers that can be usedin assessing inflammation include ESR, CRP, cytokines, chemokines, andother inflammatory mediators. ESR and CRP are measured in blood, and theother molecular markers of inflammation can be measured in blood orsynovial fluid. Use of molecular markers in blood for identifyingindividuals with pre-clinical RA or early-stage RA is described inSokolove et al. (PLoS One. 2012; 7(5):e35296) and Deane et al.(Arthritis Rheum. 2010 62(11):3161-72.).

The presence of pre-clinical and early-stage inflammatory disease may bedetermined or confirmed by a difference in level of a molecular andinflammatory markers in body fluids, including without limitationsynovial fluid, or joint tissue relative to that in a control body fluidor joint tissue from individuals free of arthritis. Examples of suchchanges in levels of molecular markers in pre-clinical and early-stageOA and RA are the following: increase in level of interleukin-1 beta(IL-1β); increase in level of TNF; increase in ratio of IL-1β to IL-1receptor antagonist protein (IRAP); increase in expression of p55 TNFreceptors (p55 TNF-R); increase in level of interleukin-6 (IL-6);increase in level of leukemia inhibitory factor (LIF); altered levels ofinsulin-like growth factor-1 (IGF-1), increase in levels of transforminggrowth factor beta (TGFβ), platelet-derived growth factor (PDGF), orbasic fibroblast growth factor (b-FGF); increase in level of keratansulfate; increase in level of stromelysin; increase in ratio ofstromelysin to tissue inhibitor of metalloproteases (TIMP); increase inlevel of osteocalcin; increased alkaline phosphatase; increased cAMPresponsive to hormone challenge; increased urokinase plasminogenactivator (uPA); increase in level of cartilage oligomeric matrixprotein; increase in level of collagenase; increase in level of othercytokines; increase in in level of CRP; or increase in level ofautoantibodies against synovial joint proteins or other biomolecules.The term “metalloprotease” as used herein is intended to refer to MMPs,especially those whose levels are typically elevated concentrationswhere there is articular cartilage degeneration, i.e., stromelysins,collagenases, and gelatinases. Aggrecanase is also included within thisterm. The three collagenases present in articular cartilage during theearly stages of degeneration are collagenase-1 (MMP-1), collagenase-2(MMP-8), and collagenase-3 (MMP-13). Of the three stromelysins,stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3(MMP-11), only stromelysin-1 appears in articular cartilage during theearly stages of its degeneration. The metalloproteases are secreted bychondrocytes as proenzymes, which must be activated before they candegrade extracellular matrix macromolecules.

A reference range for such molecular markers of inflammation is definedas the set of values within which 95 percent of the normal populationfalls. If the value or level of a molecular marker of inflammation in anindividual patient is outside the set of values or levels within which95 percent of the normal population falls, then the marker is consideredto exhibit an abnormal level in that patient (e.g. that patient isdetermined to have an “abnormal marker” or an abnormal molecular markerof inflammation).

IL-1, which exists as IL-1α and IL-1β, is a catabolic cytokine thatmediates articular cartilage injury and loss in mammalian joints. Itsuppresses the synthesis of type II collagen found in articularcartilage, while promoting the synthesis of type I collagencharacteristic of fibroblasts; induces the production of enzymesinvolved in matrix degradation; and suppresses the ability ofchondrocytes to synthesize new proteoglycans. IL-1 and its modulatorIRAP are produced in an autocrine and paracrine fashion by synovialmacrophages, and IRAP production may increase in the presence ofgranulocyte macrophage colony-stimulating factor (GM-CSF). However, IL-1is much more potent than IRAP, with approximately 130-fold more IRAPbeing required to abolish the pathogenic effects of IL-1, as measured inchondrocytes and cartilage explants. Imablances between IL-1 and IRAPexacerbates the degeneration of articular cartilage. Consequently, it isalso appropriate to identify abnormalities in the levels of IL-1 andIRAP, as well as in the ratio of IL-1 to IRAP, to identify an individualin the early stages of cartilage injury and loss before focal cartilageloss can be identified radiographically. Thus, determining the levels ofIL-1 and IRAP, as well as the ratio of IL-1 to IRAP, could enableidentification of individuals that are candidates for earlypharmacological intervention before significant cartilage degenerationoccurs. Furthermore, the frequency of IL-1α- and IL-1β-secretingmacrophages is significantly greater in the synovial fluid and synovialtissue of joints undergoing the early stages of articular cartilagedegeneration can be detected and is significantly greater than insynovial fluid and synovial tissue from normal joints, i.e., joints inwhich there is no articular cartilage degeneration.

In mammals subjected to sectioning of the cruciate ligament of a kneejoint, the concentration of TNF is statistically higher in the synovialfluid of the sectioned knee joint than in that of the contralateral,unsectioned knee joint. The expression of p55 TNF receptors (TNF-R) onchondrocytes in articular cartilage is also higher in the sectioned kneejoint. Therefore, because an increase in TNF levels, and possibly TNFsignaling, is associated with early cartilage injury and loss, it isappropriate to measure levels of TNF and TNF-R in the joints ofindividuals at risk for cartilage degeneration and loss. These resultscontribute to diagnostic classification of individuals that arecandidates for early pharmacological intervention before significantcartilage degeneration occurs.

IL-6 is an inflammatory cytokine whose levels are abnormally high(statistically elevated) in the joints and synovial fluid of damagedlimbs as compared to healthy joints and synovial fluids. IL-6 increasesthe expression of TNF-R on chondrocytes and the production ofproteoglycan by chondrocytes; it also induces the release ofglycosaminoglycans from the cartilage matrix. Comparing IL-6 levels insynovial fluid and chondrocytes of joints in the early stages ofarticular cartilage injury and loss to that in synovial fluid andchondrocytes of control joints can identify individuals that areappropriate candidates for pharmacological treatment, before any focalcartilage loss is detectable by radiographic examination.

LIF is produced by monocytes, granulocytes, T cells, fibroblasts, andother cell types associated with inflammatory conditions. Synoviocytesand chondrocytes synthesize and secrete LIF in the presence of IL-1β andTNFα. Thus, identifying increases in levels of LIF can allow selectionof candidates for pharmacologic treatment of the early stages ofarticular cartilage injury and loss.

IGF exists as types I and II, and IGF-I mediates cartilage synthesis.Furthermore, it reduces degradation and promotes synthesis ofproteoglycans even in the presence of IL-1β and TNFα. Serum levels ofIGF-1 are maintained by high-affinity binding proteins (IGF-BPs), andIGF-1 regulates both bone and cartilage turnover. Detecting abnormallyhigh levels of IGF-1 permits identification of candidates for earlypharmacologic treatment of articular cartilage degeneration.

TGFβ is produced by chondrocytes and acts as a powerful mitogencontributing to the turnover of both cartilage and bone. Further, itstimulates the synthesis of extracellular matrix and hasanti-inflammatory activity. It also inhibits the degradation of theextracellular matrix by stimulating the production of proteaseinhibitor, and blocking the release of collagenases andmetalloproteases. Further still, it promotes cartilage repair bystimulating production of collagen, fibronectin, inhibitors ofplasminogen activators, and tissue inhibitors of metalloproteases (TIMP)by various cells in the mammal joint. Synovial fluid levels of TGFβ areabnormally low in the joints of mammals in the early stages of articularcartilage injury and loss. Consequently, levels of TGFβ compared tocontrol permit diagnostic evaluation of candidates for earlypharmacologic treatment of articular cartilage degeneration.

With progressive degeneration, i.e., catabolism of the articularcartilage in the joint, a number of metabolites are produced that areuseful as markers of the cartilage degeneration, both to the occurrenceand to the progression of cartilage degeneration. For example, IL-1α andIL-1β or TNFα active inflammatory and degradative pathways that mediatecartilage degradation and release of glycosaminoglycans (GAGS), whichcan be measured in the synovial fluid of an individual. Furthermore, GAGlevels change after treatment so that it is possible to monitor theefficacy of pharmacologic intervention, by using GAG levels in synovialfluid as a marker of articular cartilage turnover. Because thedegradation of articular cartilage involves collagen as well as theother cartilage components, several collagen breakdown products serve asmarkers of cartilage degradation in mammals. Type-II-specific collagenbreakdown products, e.g., 20-30 amino acid neoepitopes, can beidentified in body fluids such as synovial fluid, plasma, serum, andurine. The presence of collagen neoepitopes in these body fluids may beused as indicators of OA onset and progression.

The presence or an increase in the levels of 5D4, a neo-epitope of theGAG keratan sulfate, in synovial fluid is a marker of early articularcartilage injury and loss. Conversely, presence of or increased levelsof various neo-epitopes of chondroitin sulfate, another GAG, isassociated with anabolic events in the articular cartilage of mammals inthe early stages of cartilage injury and loss. Levels of these epitopesin synovial fluid, particularly 3B3, 7D4 and 846, can be determined byspecific monoclonal antibodies. The 3B3 epitope is expressed onchondroitin sulfate chains of cartilage during repair and remodeling ofthe extracellular matrix, and consequently its levels in synovial fluidcorrelate inversely with those of 5D4. The determination of 3B3 levelsin the synovial fluid of test mammals and comparison of these levelswith control values permits the creation of a diagnostic profile of amammal that is an appropriate candidate for early pharmacologictreatment.

Additional markers of cartilage anabolism are the propeptides of type IIprocollagen (PIIP). Type II collagen is the major collagen of articularcartilage and is produced by chondrocytes as the procollagen PIIP.During the process of collagen fibril formation, aminopropeptide andcarboxypropeptide, the noncollagenous portions of PIIP, are cleaved andreleased into body fluids, where they can be measured as a reflection ofanabolic activity in articular cartilage. Levels of thecarboxypropeptide of PIIP (carboxy-PIIP) in synovial fluid are higherduring cartilage anabolism and correlate with radiographic evidence ofpathologic changes in the cartilage. Accordingly, detection of increasedlevels of carboxy-PIIP in synovial fluid identifies individual for earlypharmacologic treatment.

Perturbation of the stromelysin:TIMP ratio in articular cartilage andjoint fluids of mammals is another marker of early-stage articularcartilage degeneration. Abnormal joint loading after joint injury causesthe production of excess stromelysin, an enzyme produced by chondrocytesand synoviocytes in an IL-1-mediated process. The concentrations ofstromelysin are higher in fibrillated (injured) cartilage than they arein cartilage more distal to the injury. Levels of stromelysin may beexcessively high for only a short period of time, but where the damageto the joint transcends the tidemark zone of the articular cartilage andreaches into the subchondral bone, there is a substantial likelihood ofsubsequent articular cartilage degeneration, usually preceded by astiffening of the subchondral bone. In such situations, there is anincreased number of cells involved in the synthesis of stromelysin,IL-1α, IL-1β, and the oncogene proteins c-MYC, c-FOS, and c-JUN. In thesynovium cells that secrete these factors are the superficial synoviallining cells, while in the cartilage such cells are the chondrocytes inthe superficial and middle layers and the condrocytes in the fibrillatedareas of the tibial plateau. Further, stromelysin and IL-1 diffuse intothe cartilage matrix of the tibial plateau. Stromelysin, which degradescomponents of connective tissue, including proteoglycans and type IXcollagen, is actively synthesized in the synovium of mammals in theearly stages of articular cartilage degeneration, and is the primaryproteolytic enzyme involved in the cartilage destruction. Increasedlevels of stromelysin mRNA are detectable in the synovia of suchmammals, as are increased levels of collagenase mRNA. Increased levelsof both isoforms of IL-1, but especially IL-1β, stimulate the synthesisof stromelysin and collagenase by synovial fibroblasts. IL-1 does notstimulate the production of tissue inhibitor of metalloprotease (TIMP),such that the levels of this metalloprotease inhibitor in the synoviumremain unchanged while the levels of metalloproteases are dramaticallyincreased. The above text represents a detailed description is for OAand RA, but the approach, the types of markers, and a subset of themarkers are relevant for a wide spectrum of inflammatory diseases, andthese descriptions are meant to serve as an example of how oneapproaches developing markers for a pre-clinical or early-stageinflammatory diseases in general.

In some embodiments the methods of the invention comprise a step ofdetermining the presence of early-stage inflammatory disease in anindividual or susceptibility to development of inflammatory diseaseprior to treatment, and thus indicating a need of treatment. The methodmay further include determining the presence of inflammation, prior tothe administering step, where an individual at increased risk or in anearly stage of an inflammatory disease showing signs of inflammation,particularly inflammation of the relevant organ is selected fortreatment with DHCQ, or the combination therapy of DHCQ andatorvastatin. The markers relevant to each disease are presented in thedescriptions of each of the diseases. Such markers include clinicalmarkers, metabolic markers, inflammatory markers, imaging markers,research markers, and other markers, with distinct subsets of markersbeing relevant for different diseases. The markers are consideredabnormal when their levels or values or results are outside thedistribution of levels, values or results observed for 95% of thehealthy population. Individuals with abnormal levels of disease-specificinflammatory markers, metabolic markers, clinical markers, imagingmarkers, or other markers are at increased risk for developing or in theearly stages of an inflammatory disease or disease associate withinflammation.

In some embodiments, the treatment with DHCQ, or the combination of DHCQand atorvastatin, prevents the development of disease. In someembodiments the treatment with DHCQ, or the combination ofDHCQ+atorvastatin, prevents the progression of signs or symptoms of aninflammatory disease. In some embodiments the treatment with DHCQ, orthe combination of DHCQ+atorvastatin, results in the early signs orsymptoms of an inflammatory disease returning to normal. In someembodiments, treatment with DHCQ, or a combination of DHCQ andatorvastatin, results in normalization of inflammatory markers. In someembodiments the treatment with DHCQ, or the combination ofDHCQ+atorvastatin, prevents development of organ or tissue damage. Insome embodiments the treatment with DHCQ, or the combination of DHCQ andatorvastatin, results in stabilization or normalization of laboratorytest, imaging markers, or other markers of disease.

In yet other embodiments, the treatment with DHCQ, or a combination ofDHCQ and atorvastatin, is used to treat established disease in anindividual exhibiting elevated inflammatory markers (abnormalinflammatory markers). In some embodiments, treatment of establishedinflammatory disease with DHCQ, or the combination of DHCQ andatorvastatin, results in normalization of inflammatory markers and otherdisease markers. In some embodiments the treatment with DHCQ, or thecombination of DHCQ and atorvastatin, results in stabilization ornormalization of laboratory test, imaging markers, or other markers ofthe disease. In some embodiments the treatment with DHCQ, or thecombination of DHCQ and atorvastatin, prevents development of organ ortissue damage.

Various techniques and reagents can be used in the analysis ofinflammatory markers in the present invention. In one embodiment of theinvention, blood or synovial fluid samples, or samples derived fromblood, e.g. plasma, serum, etc., are assayed for the presence ofspecific biomarkers. Other sources of samples are body fluids such assynovial fluid, lymph, cerebrospinal fluid, bronchial aspirates, saliva,milk, urine, and the like. Also included are derivatives and fractionsof such cells and fluids. Diagnostic samples are collected any time thatan individual is suspected of having an inflammatory disease or of beingat risk of developing an inflammatory disease. Such assays come in manydifferent formats, including autoantigen arrays; enzyme-linkedimmunosorbent assays (ELISA) and radioimmunoassays (RIA); assays inwhich binding of labeled peptides in suspension or solution are measuredby flow cytometry or mass spectrometry.

Many such methods are known to one of skill in the art, including ELISA,fluorescence immunoassays, protein arrays, eTag system, bead-basedsystems, tag or other array-based systems, surface plasmon resonance(SPR)-based detection systems, etc. Examples of such methods are setforth in the art, including, inter alia, chip-based capillaryelectrophoresis: Colyer et al. (1997) J Chromatogr A. 781(1-2):271-6;mass spectroscopy: Petricoin et al. (2002) Lancet 359: 572-77; eTagsystems: Chan-Hui et al. (2004) Clinical Immunology 111:162-174;microparticle-enhanced nephelometric immunoassay: Montagne et al. (1992)Eur J Clin Chem Clin Biochem. 30(4):217-22; the Luminex XMAP bead-arraysystem (www.luminexcorp.com); and the like, each of which are hereinincorporated by reference.

For multiplex analysis, arrays containing one or more detectionantibodies that recognize biomarkers of interest can be generated.Various immunoassays designed to quantitate the biomarkers may be usedin screening. Measuring the concentration of the target protein or otherbiomarker in a sample or fraction thereof may be accomplished by avariety of specific assays. For example, a conventional sandwich-typeassay may be used in an array, ELISA, RIA, bead array, etc. format.

The analysis of a biological sample may be done by using any convenientprotocol, for example as described below. The readout may be a mean,average, median or the variance or other statistically or mathematicallyderived value associated with the measurement. The readout informationmay be further refined by direct comparison with the correspondingreference or control readout.

Following quantitation of the marker in the sample being assayed, thevalue obtained is compared with a reference or control value to make adiagnosis regarding the phenotype of the patient from whom the samplewas obtained. Typically a comparison is made with the analogous valueobtained from a sample or set of samples from an unaffected individual.Additionally, a reference or control value may be a value that isobtained from a sample of a patient known to have an autoimmune ordegenerative disease of interest, such as RA or OA, and therefore may bea positive reference or control profile.

For prognostic purposes, an algorithm can be used that combines theresults of determinations of multiple antibody specificities and/orcytokine levels, and/or levels of cartilage degeneration markers, and/orother markers, and that will identify individual with abnormal levels ofsuch markers and thus discriminate robustly between individuals withincreased risk for developing or with established autoimmune disease,e.g. RA, or degenerative disease, e.g. OA, and controls.

Examples of molecular markers of inflammation (also termed markers ofinflammation) include c-reactive protein (CRP), high-sensitivity CRP(hs-CRP) (or regular CRP), erythrocyte sedimentation rate (ESR), serumamyloid A, serum amyloid P, fibrinogen, cytokines in blood or otherbiological fluids, a cytokine, an antibody (such as an autoantibody, oran anti-microbial antibody), a DNA sequence, a RNA sequence (forexample, mRNA encoding one or more cytokines or other immune molecules),other markers of inflammation, or combinations thereof.

C reactive protein (CRP), including high-sensitivity CRP (hs-CRP) areincluded as a marker of inflammation and provide utility as a marker ina variety of inflammatory diseases. It is known that individuals withhigh levels of hs-CRP (abnormal levels, e.g. an abnormal inflammatorymarker), even at the high end of the normal range, have 1.5 to 4 timesincreased risk of developing an inflammatory disease or diseaseassociated with inflammation, including but not limited toatherosclerotic disease, atherosclerotic cardiovascular disease, RA,psoriatic arthritis, systemic lupus erythematosus, osteoarthritis, typeII diabetes, metabolic syndrome, NAFLD, NASH and other inflammatorymetabolic diseases. The American Heart Association and U.S. Centers forDisease Control and Prevention have defined risk groups based on hs-CRPlevels as follows:

-   -   Low risk: hs-CRP less than 1.0 mg/L    -   Average risk: hs-CRP 1.0 to 3.0 mg/L (which represents an        abnormal inflammatory marker)    -   High risk: hs-CRP above 3.0 mg/L (which represents an abnormal        inflammatory marker)

The range of levels of plasma fibrinogen that is deemed normal variesfrom laboratory to laboratory but is typically 1.5-4.0 g/L. Levels ofplasma fibrinogen above 2.8 g/L are considered an abnormal inflammatorymarker and are associated with increased risk of developing aninflammatory disease, and levels >4 g/L are associated with an evenhigher risk.

Normal levels of serum amyloid A (SAA) range widely. However, elevationsin SAA levels have been associated with increased risk of inflammatorydisease with moderate elevation (>3.9 but <8 mg/L; an abnormal SSA[inflammatory marker] level) conferring increase risk over the lowesttercile, and values greater than 8.2 mg/L (highest tercile; an abnormalSSA [inflammatory marker] level) imparting highest risk.

There is a wide range in ESR values that are considered normal, but ESRvalues that are abnormal and thus indicative of inflammation includeESR >15 mm/hr in men under 50 years old, >20 in men over 50 years oldand women under 50 years old, and >30 mm/hr in women over 50 years old.The measurement of abnormal ESR (inflammatory marker) levels in apatient indicate that that individual patient is at increased risk fordeveloping an inflammatory disease or a disease associated withinflammation.

Abnormal metabolic markers include total cholesterol (TC) greater thanabout 160 mg/dL, greater than about 180 mg/dL, greater than about 190mg/dL, greater than about 200 mg/dL, greater than about 210 mg/dL,greater than about 220 mg/dL, greater than about 230 mg/dL, greater thanabout 240 mg/dL, greater than about 250 mg/dL, greater than about 260mg/dL. Abnormal metabolic markers include an LDL cholesterol greaterthan about 70 mg/dL, greater than about 80 mg/dL, greater than about 90mg/dL, greater than about 100 mg/dL, greater than about 110 mg/dL,greater than about 120 mg/dL, greater than about 130 mg/dL, greater thanabout 140 mg/dL, greater than about 150 mg/dL, greater than about 160mg/dL. Abnormal metabolic marker levels include an HDL cholesterol lessthan about 60 mg/dL, less than about 50 mg/dL, less than about 40 mg/dL,less than about 30 mg/dL, less than about 20 mg/dL. Abnormal metabolicmarkers include an elevated LDL particle number or reduced HDL particlenumber relative to the general population. Abnormal metabolic markersare predictive for increased risk for development of, the pre-clinicalperiod of, early-stages of, or established diseases includinghyperlipidemia, atherosclerosis, atherosclerotic disease, NASH, NAFLD,metabolic syndrome, and other inflammatory diseases and diseasesassociated with inflammation.

Other inflammatory and metabolic markers of patients at risk formetabolic disease associated with inflammation include: lipoprotein a(LPa), with an abnormal metabolic marker level being greater than about30 mg/dL; Apolipoprotein A1, with an abnormal metabolic marker levelbeing less than about 123 mg/dL; Apolipoprotein B, with an abnormalmetabolic marker level being greater than about 100 mg/dL; lipoproteinassociated phospholipase A2 (Lp-PLA2), with an abnormal metabolic markerlevel being greater than about 200 ng/ml; or an urinaryalbumin/creatinine ratio, with a ratio greater than about 30 mg/grepresenting an abnormal marker ratio.

MRI, with or without gadolinium or other contrast enhancement, can beused to detect the presence of inflammation and thereby identifyindividuals with an inflammatory disease or at increased risk ofdeveloping an inflammatory disease. For example, MRI-detectedinflammation (which represents an abnormal imaging marker result) isdefined by the presence of one or more of the following findings:synovitis (synovial lining thickening, proliferation and/orenhancement), a joint effusion, bone marrow edema, and other MRI imagingfindings suggestive of inflammation (Krasnokutsky et al, ArthritisRheum. 2011 63(10):2983-91. doi: 10.1002/art.30471 PMID: 21647860;Roemer et al, Osteoarthritis Cartilage. 2010 October; 18(10):1269-74.PMID: 20691796; Guermazi et al, Ann Rheum Dis. 2011 70(5):805-11, PMID:21187293). Guermazi et al. (Guermazi et al, Ann Rheum Dis. 201170(5):805-11, PMID: 21187293) defines a semiquantiative scoring systemfor grading the level of inflammation in joints, allowing one todetermine (1) whether an individual has inflammation or not, and (2) thedegree of inflammation in an individual. Individuals with evidence ofjoint inflammation according to the Guermazi scoring system can beclassified as having increased risk for the development of OA,pre-clinical OA, early-stage OA, or established OA. The degree ofinflammation as evaluated by the Guermazi scoring system predictsdevelopment and/or progression of the inflammatory disease OA. MRI, withor without gadolinium, can be applied to many other conditions todetermine whether or not inflammation (an abnormal imaging markerresult) is present, and when present an individual is at increased riskfor developing, has pre-clinical, has early-stage inflammatory disease,or has established inflammatory disease or disease associated withinflammation.

Ultrasound-detected inflammation (an abnormal imaging marker) is definedby the presence of one or more of the following findings: synoviallining thickening and/or enhancement, a joint effusion, bone marrowenhancement, a Doppler-flow signal in the synovial lining, and otherfindings suggestive of inflammation (Guermazi et al, Curr OpinRheumatol. 2011 23(5):484-91. PMID: 21760511; Hayashi et al,Osteoarthritis Cartilage. 2012 March; 20(3):207-14. PMID: 22266236;Haugen et al, Arthritis Res Ther. 2011; 13(6):248. PMID: 22189142).Individuals with ultrasound-detected inflammation are at increased riskfor, in the early stages of, or have established inflammatory disease ordisease associated with inflammation.

In various embodiments, this invention relates to the use of DHCQ, orDHCQ in combination with atorvastatin, to treat inflammatory diseasesand diseases associated with inflammation. In one embodiment the statincomprises atorvastatin, and in other embodiments the statin can comprisecerivastatin, fluvastatin, lovastatin, mevastain, or pitavastatin.Importantly, this novel use of a combination of DHCQ and a statin doesnot require use of an antibiotic, an anti-viral, or an anti-bacterialagent. No antibiotic, anti-viral, or anti-bacterial compound is neededfor the anti-inflammatory activity and disease-modifying activitydescribed for DHCQ, or the combination of DHCQ and a statin.

In certain in vitro assays, ex vivo assays, and in vivo models, thecombination of DHCQ and atorvastatin exhibits unexpected and surprisingsynergy in reducing the production of inflammatory mediators in in vitroand ex vivo assays, and in reducing disease activity and inflammation inin vivo models. In other in vitro assays, ex vivo assays, and in vivomodels, the combination exhibits an unexpected and surprising additiveeffect in reducing the production of inflammatory mediators in in vitroand ex vivo assays, and reducing disease activity and inflammation inthe in vivo model. In general, the DHCQ alone and individual statinalone, did not provide as robust anti-inflammatory or disease-modifyingactivity as did the combinations (the combination of DHCQ+atorvastatin),which can provide for a synergistic benefit when combined.

Multiple markers of inflammatory disease, and specifically detection ofabnormal levels of these markers, can be used to identify individuals atincreased risk for disease, with early-stage disease, as well as tomonitor response to intervention with DHCQ, or DHCQ and atorvastatintherapy. Such markers, also termed biomarkers, including laboratory testresults, imaging results, physical findings, research test markers, andother markers of inflammation and disease. Examples of laboratorymarkers include: hs-CRP as a measure of systemic inflammation; ESR as ameasure of systemic inflammation; hemoglobin A1C as a measure of poorglucose control and thus the severity of diabetes and/or metabolicsyndrome; liver enzyme tests as a measure of hepatic dysfunction and ofthe activity of NAFLD or NASH; and cholesterol and LDL cholesterol as asign of atherosclerosis. Examples of imaging markers include, evidenceof early synovitis on MRI of the hand joints in individuals withpre-clinical or early stage RA; evidence of low-grade synovitis on MRIof a joint in individuals at-risk for OA; evidence of demyelinatinglesions on MRI of the brain of an individual at risk for MS. Examples ofresearch biomarkers include: multiplex profiling of cytokines in bloodto identify individuals with systemic inflammation, and to determine thespecific subset of cytokines causing the individual to be “at-risk” ormediating early-stage disease; analysis of gene expression to subtypethe inflammatory disease; analysis of genetic variants throughgenotyping or sequencing the genome of an individual to determine whichinflammatory disease(s) an individual is at increased risk fordeveloping. In other embodiments, such laboratory, imaging and researchbiomarkers are used to identify individuals at increased risk fordeveloping, or with early-stage, inflammatory disease. In otherembodiments, such laboratory, imaging and research biomarkers are usingto monitor an individual's response to DHCQ therapy, to determine iftherapy need to be continued, or if therapy needs to be increased, or ifan individuals' risk has decreased and thus therapy can be discontinued.

EXAMPLES

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

Example 1 Treatment with Desethylhydroxychloroquine (DHCQ) Prevented theDevelopment of and Reduced the Severity of Murine Rheumatoid Arthritis(RA)

DBA/1 mice (n=12-15 per group) were induced to develop collagen-inducedarthritis (CIA), a mouse model for RA, by immunization with type IIcollagen emulsified in complete Freund's adjuvant (CFA) followed 21 dayslater by boosting with type II collagen emulsified in incompleteFreund's adjuvant (IFA). On the day of the first immunization, atimepoint at which mice exhibited no symptoms of RA but had already beeninduced for and exhibit an increased inflammatory state (due to theimmunization with CFA) and develop autoantibodies by day 14 during thispre- or early-RA disease period, treatment was initiated withhydroxychloroquine (HCQ), desethylhydroxychloroquine (DHCQ),desethylchloroquine (DCQ), or bisdesethylchloroquine (BDCQ), and eachmolecule was dosed at 50 mg/kg/day by oral gavage (once daily dosing)for 2 weeks starting on the day of the initial immunization, thenincreased to a loading dose of 100 mg/kg/day by oral gavage starting onday 14 to efficiently achieve therapeutic drug concentrations in thetissues, and then at day 21 the dose was reduced back to and continuedat a lower maintenance dose of 50 mg/kg/d by oral gavage. Statisticalcomparisons of scores for the individual groups demonstrated thattreatment with HCQ or DHCQ resulted in statistically reduced developmentof disease and reduced disease activity as compared to treatment withthe vehicle (control) (# P<0.05 by the Mann Whitney U test). Incontrast, HCQ metabolites desethylchloroquine (DCQ) andbisdesethylchloroquine (BDCQ) were not associated with statisticalprotection or reduction in disease severity compared as to vehicletreated mice (by the Mann Whitney U test).

8 week old male DBA/1 mice (Jackson Laboratory) were used for generatingthe murine model of RA. Experiments were performed under protocolsapproved by the Committee of Animal Research at Stanford University andin accordance with NIH guidelines. DBA/1 mice were intradermallyimmunized with 100 μg/mouse of bovine collagen type II (Chondrex)emulsified in complete Freund's adjuvant (CFA) containing 250 μg/mouseof heat-killed Mycobacterium tuberculosis H37Ra (BD). Twenty-one daysafter immunization, mice were subcutaneously injected at the base of thetail with 100 μg/mouse of bovine CII emulsified in incomplete Freund'sadjuvant (IFA). Prior to approximately day 28, the mice exhibit nosymptoms of RA but due to the collagen immunization are in a state ofpre- or early-RA with increased levels of inflammation. Further, by day14 the immunized pre-RA mice have developed autoantibodies against typeII collagen, the autoantibody response undergoes epitope spreading, andthe mice have a persistently inflamed pre-RA disease state (ArthritisRes Ther. 2008; 10(5):R119; Finnegan et al, Autoimmunity. 201245(5):353-63). Mice start to manifest clinical RA at approximately day28, and inflammatory arthritis in the mice was evaluated by a visuallyscoring limb inflammation, measuring paw thickness, and weighingspleens. The visual scoring system was as follows: grade 0, no swellingor erythema; grade 1, mild swelling and erythema or digit inflammation;grade 2, moderate swelling and erythema confined to the region distal tothe mid-paw; grade 3, more pronounced swelling and erythema extending tothe ankle; grade 4, severe swelling, erythema, and joint rigidity of theankle, foot, and digits. Each limb was graded with a score of 0-4, witha maximum possible score of 16 for each individual mouse. Paw thicknesswas determined by measuring the thickness of both hind paws with 0- to10-mm calipers and calculating the mean of the two measurements.

On the day of the first immunization, treatment was initiated withhydroxychloroquine (HCQ), desethylhydroxychloroquine (DHCQ),desethylchloroquine (DCQ), or bisdesethylchloroquine (BDCQ), 50mg/kg/day delivered by oral gavage in 100 μL once per day for 2 weeks,then increased to a loading dose level of 100 mg/kg/day by oral gavagefor one week to more efficiently achieve therapeutic aminoquinolinelevels in the tissues, then starting at the time of boosting (day 21)the dose was reduced to the maintenance dose of 50 mg/kg/day of HCQ,DHCQ, DCQ or BDCQ by oral gavage. The purpose of the loading dose was toexpedite getting tissue levels of the dosed aminoquinolines up totherapeutic levels following the initiation of therapy. Mice in thecontrol group were treated with vehicle alone. Mice were scored for theseverity of arthritis using the visual scoring system (termed “arthritisscore”), and developed arthritis approximately one-week followingboosting (28 days following initial immunization). The arthritisseverity of the HCQ and DHCQ treated groups were statistically lowerthan the severity of arthritis in mice treated with vehicle,bisdesethylchloroquine (BDCQ), or desethylchloroquine (DCQ), P<0.05 byMann Whitney U test. FIG. 2 demonstrates that mice induced for CIA andtreated with HCQ or DHCQ exhibited reduced arthritis as measured bytotal arthritis score as compared to mice treated with BDCQ or vehicle(P<0.05 by Mann Whitney U test).

Example 2 Desethylhydroxychloroquine (DHCQ) Prevented the Development ofand Reduced the Severity of the Experimental AutoimmuneEncephalomyelitis Mouse Model for Multiple Sclerosis (MS)

Experimental autoimmune encephalomyelitis (EAE), a mouse model for MS,was induced in SJL mice (n=10 per group) by immunization withproteolipid protein peptide 139-151 (PLP 139-151) in CFA. Starting atthe time of immunization mice were treated with a loading dose of DHCQor HCQ 100 mg/kg/day by oral gavage to expedite achieving therapeutictissue levels of the dosed aminoquinoline following the initiation oftherapy, and on day 8 the dose was reduced to a maintenance dose of 50mg/kg/day. For the first approximately 10 days following the initialimmunization, the mice exhibit no symptoms of MS but are inflamed,develop autoantibodies, and are in a pre- or early-MS disease state.Starting on day 8, mice were scored daily for the severity of EAE. MannWhitney U test comparisons between the groups demonstrated thattreatment with DHCQ and HCQ both prevented development of and reducedthe severity of EAE compared to treatment with vehicle control orbisdesethylchloroquine (BDCQ) (FIG. 3). Thus, we demonstrated thatdesethylhydroxychloroquine prevented development of and reduced theseverity of the EAE mouse model of MS (*P<0.05 by Mann Whitney U test),and that the reduction inflammation was correlated with the reduction indisease severity. DHCQ demonstrated statistically superior activity,lowering EAE disease activity to a greater extent, as compared to HCQtreatment, at several timepoints (# P<0.05 by Mann Whitney U test).

Example 3 Desethylhydroxychloroquine (DHCQ) Treated Established MouseMultiple Sclerosis (MS)

Experimental autoimmune encephalomyelitis (EAE), a mouse model for MS,was induced in SJL mice (n=10 per group) by immunization withproteolipid protein peptide 139-151 (PLP 139-151) in CFA. On day 14,when mice exhibited an average clinical score of approximately 2.5,treatment was initiated with DHCQ 100 mg/kg/day or HCQ 100 mg/kg/day byoral gavage, and 8 days later the dose was reduced to 50 mg/kg/day. MannWhitney U test comparisons between the groups demonstrated thattreatment with DHCQ and HCQ both treated established EAE by reducing theseverity of EAE compared to treatment with vehicle control (*P<0.05; **P<0.01 by Mann Whitney U test) (FIG. 4A). At the termination of theexperiment, mice were sacrificed, and the brains harvested, fixed,sections histologically scored by a blinded examiner for the number ofinflammatory foci in the meninges and parenchyma using an established“Inflammatory Foci Score” (Chang et al, Recovery from EAE is associatedwith decreased survival of encephalitogenic T cells in the CNS ofB7-1/B7-2-deficient mice. European J. Immunology, 2003, 33(7):2022-32).As compared to the vehicle control treatment, treatment with HCQ reducedthe Inflammatory Foci Score in the meninges (* P<0.05 by two-tailed Ttest) and exhibited trends towards reductions in the parenchyma andtotal scores (FIG. 4B), while treatment with DHCQ statistically reducedthe Inflammatory Foci Score in the meninges, parenchyma as well as thetotal score (* P<0.05 by two-tailed T test) (FIG. 4C). Thus, DHCQpotently treated established multiple sclerosis in the EAE mouse model,and exhibited more robust therapeutic activity than treatment with HCQ.

Example 4 Desethylhydroxychloroquine (DHCQ) Prevented the Development ofand Reduced the Severity of Osteoarthritis (OA) in a Mouse Model

This example demonstrates that in mice surgically induced bydestabilization of the medial meniscus (DMM) to develop OA, treatmentwith desethylhydroxychloroquine statistically prevented and reduced theseverity of OA (P=0.03 by two-tailed T test) (FIGS. 5 and 6). C57BL6(B6) mice (n=6-10 per group) were surgically induced to develop OA bydestabilization of the medial meniscus (DMM). One week followingsurgical induction, treatment was initiated with vehicle control,hydroxychloroquine (HCQ), or desethylhydroxychloroquine (DHCQ) dosed at100 mg/kg/day by oral gavage (once per day). After 3 months, mice weresacrificed, joints harvested, joint sections cut, and tissue sectionsstained with safranin-O. An examiner blinded to treatment usedmicroscopy to score the severity of OA. The “Cartilage DegenerationScore” (also known as the “OA score”, “Severity Score” and “HistologyScore”) was determined by a blinded examiner, and the two-tailed T testused to determine if there were statistical differences in the CartilageDegeneration Scores between groups. DHCQ statistically prevented andreduced the severity of OA as assessed by the cartilage degenerationscore by two-tailed T test compared to treatment with the vehiclecontrol (P=0.03) (FIG. 5). Hydroxychloroquine did not result in astatistically signification prevention or reduction in OA severity,based on the cartilage degeneration score (FIG. 5).

The histologic sections generated at the termination of the mouse OAexperiment presented above and in FIG. 5 were subject to blinded scoringfor the “Osteophyte Score” (measure of the amount of osteophyte orectopic bone formation) and “Synovitis Score” (measure of the amount ofsynovial and joint inflammation). The scores between groups werecompared by two-tailed T test. As compared to the vehicle-treatedcontrol, DHCQ statistically prevented and reduced the severity ofcartilage degeneration, and also statistically prevented and reduced thedevelopment of osteophytes (P<0.01) and synovitis (P<0.01) (FIG. 6).

C57/BL6 (B6) mice (n=7-10 per group) were surgically induced to developOA by destabilization of the medial meniscus (DMM). Experiments wereperformed under protocols approved by the Stanford University Committeeof Animal Research and in accordance with NIH guidelines. Murine OA wasgenerated by surgically by destabilization of the DMM (Glasson, S., S.,et al., Osteoarthritis Cartilage, 15: 1061-1069 (2007)). One weekfollowing surgical induction of the DMM model, the mice walk and runnormally, the articular cartilage is intact and there is no evidence ofOA, but due to the surgical procedure the mice are in a pre- or early-OAdisease state and develop OA over the following months.

Mice were euthanized 3 months after surgery. Their stifle joints weredecalcified in EDTA solution, fixed in 4% paraformaldehyde, and embeddedin paraffin. Serial 4 micron sections were cut and stained withtoluidine blue. Scoring of arthritis in these histology sections wasdone according to a modified version of previously described compositescoring systems (Kamekura, S., et al. Osteoarthritis Cartilage 13:632-641 (2005); Bendele, A. M., J Musculoskelet Neuronal Interact., 1:363-376 (2001)). The “OA Score” was calculated as follows: cartilagedegeneration (0-4) was multiplied by the width (1=1/3, 2=2/3, and 3=3/3of surface area) of each third of the femoral-medial and tibial-medialcondyles, and the scores for the 6 regions were summed. To evaluateosteophyte (ectopic bone) formation, we scored toluidine-blue-stainedsections according to a previously described scoring system (Kamekura,S., et al. Osteoarthritis development in novel experimental mouse modelsinduced by knee joint instability. Osteoarthritis Cartilage 13, 632-641(2005)): 0, none; 1, formation of cartilage-like tissues; 2, increase ofcartilaginous matrix; 3, endochondral ossification. To evaluatesynovitis, we scored H&E-stained sections according to a previouslydescribed scoring system (Blom, A. B., et al. Synovial liningmacrophages mediate osteophyte formation during experimentalosteoarthritis. Osteoarthritis Cartilage 12, 627-635 (2004)): 0, nochanges compared to normal joints; 1, thickening of the synovial liningand some influx of inflammatory cells; 2, thickening of the synoviallining and intermediate influx of inflammatory cells; and 3, profoundthickening of the synovial lining (more than four cell layers) andmaximal observed influx of inflammatory cells. Scores for osteophyteformation and synovitis were recorded for the femoral-medial and thetibial-medial condyles on the operated side of the joint, and the scoresfor the two regions were summed and statistical comparisons performedusing a two-tailed T test.

Treatment was started 1 week after surgical induction of destabilizationof the medial meniscus (DMM), a time point at which mice were atincreased risk for the development of OA. DMM in mice is similar to adegenerative or traumatic meniscal tear in humans, which has beendemonstrated to increase the risk of humans for development of OA by5-fold. One week following induction of DMM, mice walk and run normally,are at increased risk for the development of OA but do not exhibitclassic histologic features of OA—specifically, at this time point thereis no evidence of overt cartilage loss or bone remodeling (osteophyteformation, subchondral bone remodeling). Nevertheless, 1 week followingDMM there is likely cartilage edema, proteoglycan loss, and other earlyfeatures characteristic of both murine and human OA.

Example 5 Desethylhydroxychloroquine (DHCQ) Prevented Development ofHigh Fat Diet-Induced Non-Alcoholic Steatohepatitis (NASH)

To evaluate the effect of DHCQ and HCQ on an animal model ofnon-alcoholic fatty liver disease (NAFLD) which can lead to thedevelopment of NASH, C57BL/6 mice (8 per group) were fed a high-fat“western-style” diet (60% caloric content from fat; Taconic) for 6weeks. The mice were asymptomatic throughout this time, but weredeveloping a pre- or early-disease state. The mice were treated,starting at the time of initiation of the high-fat diet, with HCQ (100mg/kg/day), DHCQ (100 mg/kg/day), or vehicle control, to prevent thedevelopment of NAFLD and NASH. After 6 weeks of treatment with DHCQ, HCQand vehicle (control) while on a high-fat diet, blood was collected andmice sacrificed for histological analysis of liver pathology. Liversfrom the mice in each treatment group were fixed, embedded, sectioned,stained with H&E, representative micrographs are presented in FIG. 7A(both 10× and 20× magnification), “liver scores” determined using anestablished scoring system for NASH (Brunt et al, Nonalcoholicsteatohepatitis: a proposal for grading and staging the histologicallesions, American J. Gastroenterology, 1999, 94(9):2467-74). Asdemonstrated in FIG. 7B, the “liver score” was statistically reduced inboth DHCQ treated (P<0.001) and HCQ treated (P<0.05) as compared tovehicle control treated mice (by two-tailed T test). As a serumlaboratory marker of NASH, serum levels of alanine aminotransferase(ALT; also known as serum glutamic pyruvate transaminase [SGPT]) andserum aspartate transaminase (AST; also known as serum glutamicoxaloacetic transaminase [SGOT]) were measured, and it was demonstratedthat DHCQ prevents and reduces abnormal elevations in the levels ofthese liver transaminases as compared to treatment with the vehiclecontrol (two-tailed T test; * P<0.05; *** P<0.001) (FIG. 7C,D). Thesestudies demonstrate that treatment with DHCQ prevented the developmentof, and reduced the severity of, NAFLD and NASH. Further, DHCQ exhibitedstatistically superior activity to HCQ in reducing the liver score, ASTand ALT (P<0.05 by two-tailed T test, not indicated on graph in FIG.7C-D).

Example 6 Desethylhydroxychloroquine (DHCQ) Treated Established High FatDiet-Induced Non-Alcoholic Steatohepatitis (NASH)

To evaluate the effect of DHCQ and HCQ on established NASH, C57BL/6 mice(8 per group) were fed a high-fat “western-style” diet (60% caloriccontent from fat; Taconic). Following initiation of the high-fat diet,after 2 weeks blood was drawn and analyzed for abnormal elevations inserum markers for NASH including AST and ALT. The AST and ALT becameabnormally elevated following the initial 2 weeks of high-fat diet, andat that time treatment was initiated with HCQ (10 mg/kg/day), DHCQ (10mg/kg/day), or vehicle control. After 6 weeks of treatment, mice werefasted and blood was collected for serum analysis and mice sacrificedfor histologic analysis of liver pathology. FIG. 8A presents micrographsof representative liver tissue sections at both 10× and 20×magnification. The liver sections were scored for “steatosis percentageof whole area” using a refined and adapted version of a previouslydescribed scoring system (Tous et al., Feeding apolipoprotein E-knockoutmice with cholesterol and fat enriched diets may be a model ofnon-alcoholic steatohepatitis. Mol cell Biochemistry, 2005, 268:53-59;Tous et al, Dietary cholesterol and differential monocytechemoattractant protein-1 gene expression in aorta and liver of apoE-deficient mice. Biochemical and Biophysical Research Communications,2006, 340:1078-1084), and treatment with either DHCQ or HCQstatistically reduced the steatosis percentage as assess by liverhistology (* P<0.05, ***P<0.01 by 2-tailed T test) (FIG. 8B). AST andALT levels in the collected serum were measured and 2-tailed T Testsused to compare levels between groups. Treatment with DHCQ statisticallyreduced abnormal elevations in AST and ALT as compared to vehiclecontrol (P<0.001; FIG. 8C,D), while treatment with HCQ only exhibited atrend towards reduction in abnormal elevations in AST and ALT(N.S.=non-significant). DHCQ treatment exhibited statistical reductionsin the AST and ALT as compared to treatment with HCQ (*P<0.05, **P<0.01by two-tailed T test). These studies demonstrate that DHCQ exhibitedrobust efficacy in treating established NASH.

Example 7 DHCQ Treated Type II Diabetes, Hyperlipidemia and MetabolicSyndrome in Mouse Diet-Induced Obesity

To evaluate treatment with DHCQ and HCQ on a mouse model of diet-inducedtype II diabetes, hyperlipidemia and metabolic syndrome, C57BL/6 mice (8per group) were fed a high-fat “western-style” diet (60% caloric contentfrom fat; Taconic). In an analogous fashion to that in Example 6, twoweeks following initiation of the high-fat diet groups of mice wereinitiated on treatment with vehicle control, HCQ (10 mg/kg/day) or theHCQ metabolites DHCQ (10 mg/kg/day), DCQ (10 mg/kg/day), or BDCQ (10mg/kg/day). After 6 weeks of treatment, blood was collected for analysisof glucose, triglyceride, and cholesterol levels. FIG. 9 shows graphscomparing glucose, triglyceride, and cholesterol levels for HCQ, DHCQ,DCQ, and BDCQ treatment of type II diabetes, hyperlipidemia andmetabolic syndrome in mouse diet-induced obesity. Levels of glucoserepresent a biomarker of early insulin resistance and early-onset oftype II diabetes, and treatment with DHCQ resulted in statisticalreduction in blood glucose levels as compared to treatment with vehiclecontrol (* P<0.05, two-tailed T test) (FIG. 9A), while in contrast HCQ,DCQ and BDCQ did not result in statistical reductions in blood glucoseas compared to the vehicle control (FIG. 9A). The levels of lipids werealso measured in the collected sera, and based on statistical analysisby two-tailed T test DHCQ treatment resulted in statistical reductionsin total cholesterol (***P<0.001; FIG. 9B) and triglycerides(***P<0.001; FIG. 9C), as compared to treatment with the vehiclecontrol. Treatment with HCQ, DCQ and BDCQ did not result in statisticalreductions in glucose, cholesterol or triglycerides, as compared to thelevels in vehicle control treated mice (N.S.=non-significant bytwo-tailed T test) (FIG. 9). Further, DHCQ treatment resulted instatistically significant reductions in cholesterol and triglycerides ascompared to treatment with HCQ (***P<0.001 by two-tailed T test). DHCQtreatment also resulted in statistically significant reductions inglucose, cholesterol and triglycerides as compared to treatment with DCQ(*P<0.05, ***P<0.001 by two-tailed T test), and a statisticallysignificant reduction in cholesterol as compared to treatment with BDCQ(***P<0.001 by two-tailed T test). These data demonstrate that DHCQtreated diet-induced obesity associated development of insulinresistance, type II diabetes, hyperlipidemia, and metabolic syndrome,and did so statistically more effectively than HCQ, DCQ and BDCQ.

Example 8 Desethylhydroxychloroquine (DHCQ) Reduced InflammatoryCytokine Production in Response to a Proinflammatory Stimulus

DHCQ reduced the production of the pro-inflammatory cytokine tumornecrosis factor (TNF) by human peripheral blood mononuclear cells(PBMCs) in response to pro-inflammatory lipopolysaccharide (LPS)stimulation (FIG. 10). Human PBMCs were isolated using Ficoll, 200,000PBMCs were added to each well of a 96-well plate, and stimulated withLPS 10 μg/ml in the presence of a range of concentrations from 0 to 50 MDHCQ, or 0 to 50 μM HCQ, for 14 hours, following which culturesupernatants were collected and TNF measured by ELISA. Assays were runin triplicate. Mean TNF levels with standard error of the mean aredisplayed. The Tukey test was used to statistically compare resultsbetween groups, and both HCQ and DHCQ reduced PMBC TNF production at 10,25 and 50 μM concentrations as compared to LPS stimulation in theabsence of these molecules (** P<0.001, by two-tailed T test).

Isolation of human PBMCs and monocytes. The Ficoll-Paque™ Plus (Cat;17-1440-03GE Healthcare) was used to isolate human peripheral bloodmononuclear cells (PBMCs) from blood collected from healthy donors. Forcell culture, the isolated PBMCs were resuspended in culture medium(RPMI1640) containing 10% FCS and antibiotics (penicillin 100 IU/mL andstreptomycin 100 μg/mL). Monocyte Isolation Kit II (Cat; 130-091-153,Miltenyi Biotec) was used to isolate monocytes from the suspension ofhuman PBMCs.

Stimulation assays. Human PBMCs plated at 1.0×106 cells/well in 48-wellculture plates were pretreated with HCQ or desethylhydroxychloroquine orvehicle for 60 min at 37° C., 5% CO₂. Ten micrograms per ml oflipopolysaccharide (LPS; Sigma) 20 h, at 37° C., 5% CO₂. Human monocytesplated at 5.0×104 cells/well in 96-well culture plates were pretreatedwith HCQ or desethylhydroxychloroquine or vehicle for 60 min at 37° C.,5% CO₂ and then stimulated with LPS (Sigma) for 15 h at 37° C., 5% C002.

Readout. Output from the PMBC cellular assay was TNF for the LPSstimulation assays. For each assay, a parallel well treated identicallywas prepared and level of LDH measured to assure no evidence of celldeath was induced by drug treatment.

Example 9 Desethylhydroxychloroquine (DHCQ) Treatment is Less Cytotoxicto Retinal Cells, as Compared to Treatment with Hydroxychloroquine (HCQ)or Bisdesethyhydroxychloroquine (BDCQ) which Both Resulted in IncreasedRetinal Cell Death

The major serious risk to long term administration of hydroxychloroquineis retinal accumulation and subsequent ocular toxicity (Terahi et al,2008. Semin Opthal. (3):201-208. PMID). The ocular deposition of HCQ,DHCQ, and BDCQ was evaluated by direct retinal cellular toxicity byincubating equivalent concentrations of these molecules with the retinalpigmented epithelial cell line ARPE-19. ARPE-19 is a human retinalpigment epithelial cell line with differentiated properties (Dunn etal., Exp Eye Res. 1996 62(2):155-69). ARPE-19 cells were grown to 90%confluence, and then exposed to 10 hg/ml of HCQ, DHCQ, BDCQ, or vehiclecontrol for 24 hours, following which microscopic analysis andphotomicroscopy was performed at 40× power. FIG. 11 presentsrepresentative photomicrographs demonstrating that DHCQ treatment wasassociated with less ARPE-19 cellular toxicity as compared to treatmentwith HCQ or BDCQ which resulted in increased cytotoxicity and death.Retinal epithelial cell viability was high in both the vehicle controland DHCQ treated cells. Retinal cell dysmorphophic features and retinalcell death were observed in the HCQ and BDCQ treated cells (red arrows).Thus, DHCQ exhibited less retinal cell cytotoxicity as compared to theincreased retinal cell cytoxicity observed with HCQ or BDCQ treatment.

In FIG. 12, this cytotoxicity is quantitated based on lactatedehydrogenase (LDH) release. As described above, ARPE-19 cells weregrown to 90% confluence then exposed to 10 μg/ml HCQ, DHCQ, BDCQ, orvehicle control for 24 hours followed by quantitation of cell death bylactate dehydrogenase (LDH) release assay (Abcam). FIG. 12 demonstratesthat treatment with DHCQ did not cause cellular cytotoxicity and deathas compared to treatment with vehicle control (P=N.S. (non-significant),by two-tailed T test). In contrast, both BDCQ and HCQ resulted instatistically increased LDH release and thus cellular cytotoxicity ofthe retinal epithelial cell line as compared to vehicle control (***P<0.01 by two-tailed T test). Further, equimolar concentrations of HCQand BDCQ resulted in significantly increased retinal cell toxicity ascompared to DHCQ (### P<0.001 by two-tailed T-test) (FIG. 12). Thesedata demonstrate that DHCQ exhibits minimal retinal cell toxicity invitro, which is in contrast to HCQ and the other HCQ metabolite BDCQwhich both exhibited significantly increased retinal cell toxicity.

Example 10 Demonstration that DHCQ Accumulates at Increased Levels inthe Plasma and Synovial Tissue, Relative to the Low Levels thatAccumulate in the Retina; while in Contrast HCQ Accumulates at HighLevels in the Retina Relative to its Levels in Plasma or Synovial Tissue

The major risk to long term administration of hydroxychloroquine isretinal accumulation and subsequent ocular toxicity (Terahi et al, 2008.Semin Opthal. (3):201-208. PMID). We sought to evaluate the retinaldeposition of HCQ and DHCQ. To do so, we performed mass spectrometricanalysis of drug and metabolite content in retina, in operated side andnon-operated side synovial tissue, and in plasma derived from micetreated with either HCQ or DHCQ. FIG. 13 presents the measuredconcentrations of HCQ levels in the indicated tissues (plasma, retina,synovial tissue from surgical side, synovial tissue from unoperatedside) from HCQ-dosed mice; DHCQ level in the indicated tissues fromHCQ-dosed mice; and DHCQ levels in the indicated tissues from DHCQ-dosedmice. The indicated statistical comparisons between the levels measuredin the indicated tissue from each group were compared by two-tailed Ttest (*P<0.05, **P<0.01, ***P<0.001). Comparisons of the ratios of theconcentrations of HCQ and DHCQ measured in the indicated tissues in thegroups of HCQ or DHCQ dosed mice are presented in FIGS. 14-16.

DHCQ accumulated at high levels in the plasma and synovial tissue, withonly low-level accumulation in the retina; which is in contrast to HCQwhich accumulated a high levels in the retina relative to the lowerlevels in plasma or synovial tissue (* P<0.05, *** P<0.01, by two-tailedT test) (FIG. 13). Demonstrated are increased levels of HCQ in theretina in HCQ treated mice, but only low-level accumulation of DHCQ inthe retinas of HCQ mice or in mice treated with DHCQ (* P<0.05, ***P<0.01, by two-tailed T test) (FIG. 13). Thus, in contrast to HCQ, DHCQexhibits reduced retinal accumulation in both HCQ and DHCQ treated mice.

This is further demonstrated by comparison of the ratios of levels ofDHCQ or HCQ in various dosing groups and tissues (FIGS. 14-16).Specifically, the concentrations of DHCQ and HCQ measured in the HCQ andDHCQ dosing groups in FIG. 13 are displayed as one of the 3 followingratios: (1) level in synovial tissue of operated stifle joint[knee]/level in plasma; (2) level in retina/level in plasma; (3) levelin synovial tissue of operated stifle joint [knee]/level in retina(FIGS. 14-16).

DHCQ levels were lower in the retina relative to the plasma, while incontrast HCQ accumulated at higher levels in the retina relative to theplasma (FIGS. 14 and 15). FIG. 15 presents the ratio of the level inretina/level in plasma of DHCQ in DHCQ or HCQ dosed mice, and the ratioof the level in retina/level in plasma of DHCQ in DHCQ dosed mice. Thesedata show that HCQ achieves approximately 8.5× higher levels in theretina than the plasma, while in contrast DHCQ exhibits the oppositewith much lower levels in plasma as compared to the retina in both HCQand DHCQ dosed mice.

Further, DHCQ accumulated at higher levels in the surgical jointsynovial tissue relative to the low levels measured in the retina, whilein contrast HCQ accumulated at high levels in the retina relative to thelow levels measured in the surgical joint synovial tissue (FIGS. 14 and16). FIG. 16 displays the ratio of the level in surgical-sidesynovium/level in retina of DHCQ in DHCQ or HCQ dosed mice, and theratio of the level in level in the surgical-side synovium/retina of DHCQin DHCQ dosed mice. These data show that in both HCQ and DHCQ dosedmice, that DHCQ differentially accumulated in the surgical jointsynovial tissue at high levels relative to the low levels thataccumulated in the retina. In contrast, in HCQ dosed mice, levels of HCQwere higher in the retina relative to levels in the plasma or surgicaljoint tissue. Thus, DHCQ's preferential accumulation at high levels inthe synovium of the surgical joint relative to the low levels in theretina, likely contribute to DHCQ being less likely than HCQ to causeretinal toxicity.

Mice, dosing with HCQ and DHCQ, isolation of specific tissues, and massspectrometry measurement of HCQ and DHCQ levels in tissue samples.C57BL6 (B6) mice (n=5 per group) were treated with HCQ (100 mg/kg/day)or DHCQ (100 mg/kg/day) by oral gavage for 3 months. At experimentaltermination (3 months) mice were sacrificed and eyes removed underdissecting microscope. Curved microdisecting scissors was used to cutalong the cornea and sclera, remove and discard the lens and visceralleaving the neural retina with eye shell to be placed in PBS andhomogenized followed by centrifugation. Synovium was microdissected fromthe operated knee or the contralateral non-operated knee, normalized byweight of tissue, and placed in HPLC grade water before beinghomogenized and centrifuged. Plasma was obtained by tail bleeding.Plasma and tissue samples were precipitated with acetonitrile and levelsof HCQ, DHCQ, BDCQ were evaluated by liquid chromatography/massspectrometry (LC/MS) at Climax Laboratories, Inc. (San Jose, Calif.).The LC/MS analysis was conducted by using Shimazu 10A HPLC system(Shimadzu Scientific Instruments, Inc., Pleasanton, Calif.) with ACEC18, 50×2.1 HPLC column and ABSciex API-4000 Mass Spectrometer (ABSciexCorp. Foster City, Calif.) with Electrospray Ionization (ESI) andnegative Multiple Reaction Monitoring (MRM) Scan. A gradient elution wasused in separating the test compound with a mobile phase A (0.1% FormicAcid in 5 mM of NH4AC) and B (0.1% Formic acid in acetonitrile).

Example 11 Analysis of Retinal Histology Demonstrates that In VivoTreatment with DHCQ is Associated with Less Retinal Toxicity and CellDeath as Compared to Treatment with HCQ

From groups of mice (n=5) dosed with HCQ 100 mg/kg/day or DHCQ 100mg/kg/day for 3 months as described in Example 10 and FIGS. 13-16,analysis of the retina was performed and demonstrated reduced retinaltoxicity in mice treated with DHCQ as compared to mice treated with HCQ.At the time of termination, the eyes from each treatment group werecarefully microdissected to ensure the retina remained intact, the eyewas fixed in formalin, and the fixed eye sectioned to visualize theretina. The retinal cell layer was stained with hematoxylin and eosin(H&E), and evaluated for number of nuclei in the ganglion cell layer(GCL), as well as nuclear shrinkage in the GCL which is suggestive of aselective loss of retinal ganglion cells. Based on the result presentedin FIG. 17, increased nuclear shrinkage in the GCL was observed in micetreated with HCQ (FIG. 17B). No increase in nuclear shrinkage in the GCLwas observed in the groups of mice treated with DHCQ (FIG. 17C).Representative images of H&E stained retinal sections are presented fromeach treatment group in FIG. 17.

Using the histologic and quantitative pathology methodology adapted fromShichiri, et al (Shichiri, et al, JBC. 2012, 287(4):2926-34. PMID22147702), the H&E stained retinal sections were evaluated for number ofnuclei in the ganglion cell layer (GCL) (FIG. 18). The graph presentsquantitation of the number of nuclei in the GCL of the retina from theindicated treatment groups. The number of cells in the GCL of the retinafor each treatment group was compared with the number of cells invehicle-treated control by two-tailed T test. As compared to the retinasfrom vehicle-treated control mice, we found that the number of cells inthe GCL was significantly lower in mice treated with HCQ as compared tomice treated with vehicle (* P<0.05) (FIG. 18). In contrast, as comparedto retinas from vehicle-treated control mice, there was no reduction inthe number of cells in the GCL in retinas from mice treated with DHCQ(N.S.=non-significant) (FIG. 18). Further, we statistically compared thenumber of cells in the GCL in retinas from DHCQ treated as compared toHCQ treated mice, and demonstrated that HCQ treatment resultedstatistically increased retinal cell loss as compared to DHCQ treatment(# P<0.05) (FIG. 18).

These results demonstrate that treatment with DHCQ results instatistically less retinal toxicity (retinopathy) as compared totreatment with HCQ.

Example 12 Treatment with DHCQ or the Combination of DHCQ+AtorvastatinPrevented the Development of and Reduced the Severity of MurineOsteoarthritis (OA)

FIG. 19 presents the results comparing cartilage degeneration scores forthe groups of mice treated with atorvastatin, HCQ, DHCQ, BDCQ, DCQ,atorvastatin+HCQ, atorvastatin+DHCQ, or atorvastatin+BDCQ. Thecombinations of DHCQ+atorvastatin and HCQ+atorvastatin prevented thedevelopment of and reduced the severity of osteoarthritis (OA) in amouse model. C57BL6 (B6) mice (n=7-10 per group) were surgically inducedto develop OA by destabilization of the medial meniscus (DMM). One weekfollowing surgical induction, a timepoint at which the mice wereasymptomatic or exhibit mild pre-OA joint symptoms, treatment wasinitiated with one or more of the following molecules: atorvastatin 40mg/kg/day, HCQ (HCQ) 100 mg/kg/day, DHCQ 100 mg/kg/day,desethylchloroquine (DCQ) 100 mg/kg/day, orbisdesethylhydroxychloroquine (BDCQ) 100 mg/kg/day, as individual orcombinations of molecules (as labeled in the figure), all delivered byoral gavage once per day. After 3 months, mice were sacrificed, jointsharvested, joint sections cut, and tissue sections stained withsafranin-O. The mean “Cartilage degeneration scores” in safranin-Ostained sections of the medial region of stifle joints are presented inthe graph. Two-tailed T tests were used to compare the CartilageDegeneration Scores for each group as compared to the vehicle controlgroup.

FIG. 20 is a table comparing the “cartilage degeneration scores”,“osteophyte scores”, and “synovitis scores” (for a description of thesescores, see Wang et al, Identification of a critical role for complementin osteoarthritis. Nature Medicine, 2011, 17(12):1674-9) for the groupsof mice treated with atorvastatin, HCQ, DHCQ, BDCQ, DCQ,HCQ+atorvastatin, DHCQ+atorvastatin, or BDCQ+atorvastatin. Thecombinations of DHCQ+atorvastatin, and HCQ+atorvastatin, prevented thedevelopment of OA and reduced the severity of the Cartilage DegenerationScore in a mouse model of OA. From the mouse OA experiment presented inFIG. 19, the mean “Cartilage degeneration scores” in safranin-O stainedsections of the medial region of stifle joints were compared between thevehicle treated group and each of the other treatment groups bytwo-tailed T tests, and it was demonstrated that the combination ofDHCQ+atorvastatin, as well as HCQ+atorvastatin, both statisticallyreduced synovitis (inflammation) in the joint (P<0.01), prevented thedevelopment of OA (P<0.01), and reduced the severity of OA (P<0.01) ascompared to vehicle-treated mice

FIG. 21 is a table comparing the cartilage degeneration scores,osteophyte scores, and synovitis scores for subjects treated withcombinations of HCQ, DHCQ, BDCQ, or DCQ with atorvastatin, versusmonotherapies with HCQ, DHCQ, or atorvastatin alone. Protective effectof DHCQ+atorvastatin, and HCQ+atorvastatin, compared with either HCQ oratorvastatin monotherapy alone in murine OA. From the mouse OAexperiment presented in FIG. 19, the mean “Cartilage degenerationscores”, “Osteophyte scores”, and “Synovitis scores” were comparedbetween the individual single drug treated groups (e.g.atorvastatin-treated, or HCQ-treated, or DHCQ-treated) and each of thecombination-treated groups (e.g. DHCQ+atorvastatin, or HCQ+atorvastatin)by two-tailed T tests, and it was demonstrated that the combination ofDHCQ+atorvastatin as well as HCQ+atorvastatin both statistically reducedsynovitis (inflammation) in the OA joint (P<0.01), prevented thedevelopment of OA (P<0.01), and reduced the severity of OA (P<0.01) ascompared to vehicle-treated mice.

Treatment with DHCQ alone reduced the “Cartilage Degeneration Score” ascompared to treatment with vehicle (P=0.03 by two-tailed T test) (FIGS.19 and 20). In contrast, treatment with either HCQ alone or atorvastatinalone only resulted in trends towards improvement in the “CartilageDegeneration Score” (FIGS. 19 and 20). The combination ofDHCQ+atorvastatin statistically reduced the “Cartilage DegenerationScore” by two-tailed T test both as compared the vehicle treated group(P<0.01), as well as compared to treatment with either DHCQ alone oratorvastatin alone (P<0.05) (FIG. 21). In addition, treatment with DHCQalone resulted in statistically significant protection against thedevelopment of synovitis and osteophytes as compared to treatment withvehicle (FIG. 20), and the combination of DHCQ+atorvastatin providedeven more powerful and statistically significant prevention andreduction in the development of synovitis and osteophytes as compared totreatment with either drug along (FIG. 21).

Thus, it is demonstrated that DHCQ, or a combination ofDHCQ+atorvastatin, prevented development of and reduced the severity ofosteoarthritis.

Example 13 DHCQ, the Combination of DHCQ+Atorvastatin, and theCombination of HCQ+Atorvastatin, Inhibit Development of InflammatoryCytokine Production in Synovium in a Mouse Model of OA

From a parallel experiment to the mouse OA experiment presented inExample 12 and FIG. 19, following in vivo dosing and at the time oftermination OA synovial tissue was microdissected, homogenized to form alysate, centrifuged, and supernatants assayed for levels of inflammatorycytokines using a multiplex bead-based cytokine assay (BioRadLaboratories, Hercules, Calif.). FIG. 22 demonstrates a heat maprepresenting levels of inflammatory cytokines in mouse OA synovium (SNY)derived from vehicle treated (CTRL), hydroxychloroquine treated mice(HCQ), desethylhydroxychloroquine treated mice (DHCQ), atorvastatintreated mice (Atorva), mice treated with the combination of HCQ+Atorva,and mice treated with the combination of DHCQ+Atorva. The results of twoindependent measurements from each of two independent mice from eachtreatment group are presented and analyzed. Compared with vehiclecontrol-treated mice, levels of inflammatory cytokines in OA synoviumwere lower in mice treated with HCQ alone or DHCQ alone, and were lowerstill in those treated with a combination of HCQ+atorvastatin, and werelowest in mice treated with a combination of DHCQ+atorvastatin (FIG.22). Similarly, FIGS. 22 and 23 demonstrate lower levels of multipleinflammatory cytokines in mouse OA synovial tissue from mice treatedwith DHCQ alone as compared to treatment with vehicle control (FIGS. 22and 23 A-E), as well as in mice treated with DHCQ+atorvastatin ascompared to treatment with vehicle control (FIGS. 22 and 23 F-I).Treatment with the combination of HCQ+atorvastatin also inhibited theproduction of inflammatory cytokines as compared to treatment withvehicle control (FIGS. 22 and 23J). FIG. 24 demonstrates highdimensionality analysis of multiple cytokines using the SignificanceAnalysis of Microarrays algorithm and software (Tusher, et al., PNAS.2001 98(9):5116-21. PMID 11309499) to identify cytokines differentiallyproduced in OA synovium from various treatment groups as identified by afalse discovery rate <0.1% (q-value). As demonstrated in FIG. 24:

Compared with HCQ monotherapy, treatment with the combination ofHCQ+atorvastatin resulted in significantly lower levels of MCP-1 (FIG.24 I).

Compared with atorvastatin monotherapy, the combination ofHCQ+atorvastatin resulted in significantly lower levels of TNFα andMCP1.

Compared with vehicle control treatment, treatment with the combinationof DHCQ+atorvastatin resulted in significantly lower levels of IL-1β,MCP-1, IL12p40, and IL-12p70.

Compared with atorvastatin monotherapy, treatment with the combinationof DHCQ+atorvastatin resulted in significantly lower levels of IL-1β,MCP-1, IL12p40, and IL-10.

DHCQ monotherapy reduced the levels of multiple inflammatory cytokinesincluding IL-1b, MCP-1, IFNγ, TNF-alpha IL12p40, and IL-12p70 ascompared to treatment with vehicle control, atorvastatin alone (FIG. 24H), HCQ+atorvastatin (FIG. 24 G), and HCQ alone (FIG. 24 F).

Example 14 A Combination of HCQ+Atorvastatin Results in a Reduction ofSynovitis and Improvement in Pain and Functional Scores in HumanSubjects with Medial-Compartment Knee OA in a 16-Week Open-Label PilotClinical Trial (NCT01645176)

Nearly 27 million people in the U.S. have some form of osteoarthritis(OA) which has increased from 21 million in 1990. Knee OA is prevalentin 16% of all adults 45 years and older. In Canada, OA affects 10% ofthe entire population. In 2005, the estimated annual lost productivetime of U.S. workers due to OA exceeded $70 billion. A population basedstudy conducted from 1990 to 2000 showed the incidence of total kneereplacements in patients aged >45 years increased 81.5% during thatperiod. Total charges for total knee replacements to the US healthcaresystem in 2000 were approximately $148 million.

Medical therapies used to treat OA include non-steroidalanti-inflammatory drugs (NSAIDs), acetaminophen, intraarticularcorticosteroids, intraarticular hyaluronic acid formulations, narcotics,and physical therapy. While all of these may alleviate the symptomsassociated with OA, there are no medical therapies currently availablewhich prevent the progression of cartilage loss or reverse the diseaseprocess. In patients with more severe knee OA, total joint replacementsurgery is an option. The incidence of total knee replacement issteadily rising and OA is the leading cause of knee replacement surgery.The increased incidence of knee replacement surgery is putting a burdenon the healthcare system as well as creating a risk for surgicalcomplications.

Preclinical studies demonstrated that Arthrostatin, a combination ofHCQ+atorvastatin, prevents the development of OA in the destabilizationof the medial meniscus (DMM) mouse model (FIGS. 19-21). The combinationof HCQ+atorvastatin provided statistically significant benefit in thismodel, while several other combinations and treatment with HCQ oratorvastatin alone did not result in a statistically significantreduction in the severity of OA.

To date, HCQ has been tested in human OA and exhibited trends towardstherapeutic benefit in case series in erosive OA, but has notdemonstrated disease-modifying or pain-reducing activity in non-erosiveOA.

A primary objective of the clinical trial is evaluation of the efficacyof Arthrostatin for the treatment of osteoarthritis (non-erosive)measuring differences in change synovitis of the knee between 0 and 24weeks as measured by MRI in patients with OA. A secondary objective isevaluation of the safety and tolerability of study agent over 24 weeksand evaluation of the impact of the study agent on pain and functionover 24 weeks. Exploratory objectives are ultrasound assessment ofsynovitis; and marker analysis, including markers of cartilagebreakdown, metabolism and inflammation. To date, 7 human medialcompartment knee OA patients have completed 16 weeks of dosing,including all baseline, in-life and follow-up examinations, tests andgadolinium-enhanced MRI imaging studies.

The primary endpoint for this study is determination of the proportionof subjects treated with Athrostatin achieving meaningful improvement insynovitis based on a reduction in the synovitis score (Guermazi et al.,Ann Rheum Dis. 2011 70(5):805-11. PMID: 21187293) as measured by Gd-MRIby greater than 4 points at 24 weeks in patients treated withArthrostatin as measured by MRI at 24 week. The overriding hypothesisfor this pilot trial is that interventions that reduce the low-gradesynovitis OA (as measured by Gd-MRI) in this open-label pilot trial,will provide chondroprotective effects and reduce the progression of OAin subsequent Phase II and Phase III clinical trials.

The Secondary Endpoints include the safety and tolerability ofArthrostatin in subjects with early OA; change from baseline to Weeks 4,12 and 24 in the WOMAC pain subscale and change from baseline to Weeks4, 12 and 24 in the WOMAC function subscale; change from baseline toWeeks 4, 12 and 24 in the Patient's Global VAS; analysis of efficacydata using the OMERACT-OARSI Responder Index (Onel et al, Clin DrugInvestig. 2008; 28(1):37-45. PMID: 18081359); change from baseline toWeeks 4, 12 and 24 weeks in HAQ-DI; change from baseline to Weeks 4, 12and 24 in the Physician's Global VAS; and to determine the use of rescuemedications required at 4, 12 and 24 weeks.

Subjects with OA were recruited and informed consent was obtained.During a screening period lasting up to 34 days, subjects underwentmedical and arthritis history, physical examination, and complete theWOMAC pain and function subscale questionnaires and patient VAS globalassessment. ECG, bilateral knee x-rays and MRI of the index knee isperformed and concomitant medications are recorded. Samples wereobtained for urinalysis, hematology, blood chemistry, and a urinepregnancy test (for women of childbearing potential). Vital signs andweight are recorded. Subjects are asked to maintain their usual dose ofNSAIDs and/or other analgesics during the course of the trial, exceptduring the 48 hour period or 24 hour for acetaminophen precedingefficacy assessments (WOMAC and HAQ questionnaires, and Patient GlobalAssessment VAS) at Day 1 (baseline), and Weeks 2, 4, 12 and 24.

Subjects who met all of the inclusion criteria and none of the exclusioncriteria were entered into the study on Day 1 and will receiveArthrostatin. Additional follow-up visits are conducted at Weeks 2, 4,12 and 24 and safety and efficacy assessments performed according to theSchedule of Assessments. Telephone follow-up visits will occur at Weeks8, 16, and 20. The dosing regimen is HCQ 400 mg/d and atorvastatin 40mg/d.

Inclusion Criteria (abnormal markers): 1. Ambulatory subjects with OA ofthe knee with symptoms for at least 6 months and pain on the majority ofdays in the last 30 days (assessment of abnormal clinical markers).Symptoms must include knee joint pain. In subjects with bilateral kneeOA, the more symptomatic knee is the index knee (assessment of abnormalclinical markers). 2. Male or female adults age >40 years with a bodymass index <35 (measurement of abnormal metabolic marker). 3.Radiographic evidence of at least one osteophyte in either knee onposteroanterior (PA) and lateral standing, flexed x-ray (measurement ofabnormal imaging marker). 4. An OARSI Atlas joint space narrowing gradeof 1 or 2 in the index knee (imaging of abnormal imaging marker). 5. AWOMAC pain score of >8 on the index knee at screening visit 2 and at Day1/baseline visit (measurement of abnormal clinical marker). 6. Asynovitis score of 9-14 based on gadolinium-enhanced MRI (Gd-MRI) of theindex knee and the scoring system (based on summed scores from 11 sites)described in Guermazi et al (Ann Rheum Dis. 2011 70(5):805-11. PMID:21187293) (measurement of abnormal imaging marker). 7. Able to complywith the study and give informed consent. 8. Able to read, write andunderstand English.

For this trial, candidate patients were assessed for evidence of thelow-grade inflammatory disease OA based on the required presence ofmultiple abnormal clinical and laboratory markers. Clinical markersmeasured and required for trial entry included knee pain for at least 6months, knee pain localizing to one side, and a WOMAC pain score >8. Inaddition, two imaging markers were required for trial entry includingradiographic evidence of at least one osteophyte in either knee onposteroanterior (PA) and lateral standing flexed x-ray and a synovitisscore of 9-14 based on gadolinium-enhanced MRI (Gd-MRI) of the indexknee and the scoring system (based on summed scores from 11 sites)described in Guermazi et al (Ann Rheum Dis. 2011 70(5):805-11. PMID:21187293) (measurement of abnormal imaging marker). Based on themeasurement and detection of these abnormal clinical and abnormalimaging markers (as described in detail in the inclusion criteria),individuals were enrolled in the pilot trial and treated with thecombination of HCQ+atorvastatin.

Exclusion Criteria: 1. A requirement for treatment with high potencyopioids for pain relief. 2. Unwilling to abstain from NSAIDs and/orother analgesic medications except acetaminophen (i.e., COX-2inhibitors, tramadol) for 48 hours and acetaminophen for 24 hours priorto pain assessments during the study. Subjects taking low dose aspirinfor cardiovascular health may remain on their stable dose throughout thestudy. 3. On an unstable dose of NSAIDs or analgesics for at least 3months prior to screening visit 1. 4. Using a handicap assistance device(i.e., cane, walker) >50% of the time. 5. Undergoing new physicaltherapy or participating in a weight loss or exercise program that hasnot been stable for at least 3 months prior to screening visit 1 andwill not remain stable during their participation in the study. 6. Had aprevious history of arthroscopic or open surgery to the index knee inthe past 6 months or planned surgery during study follow up. 7. Hadjoint replacement surgery in the index knee. 8. Received corticosteroid,short acting hyaluronic acid, or other intraarticular injections of theindex knee within 3 months of screening visit 1 and/or not willing toabstain from treatments for the duration of the study. 9. A history inthe past 5-10 years of reactive arthritis, rheumatoid arthritis,psoriatic arthritis, ankylosing spondylitis, arthritis associated withinflammatory bowel disease, sarcoidosis, amyloidosis or fibromyalgia.10. Clinical signs and symptoms of active knee infection or radiographicevidence of crystal disease other than chondrocalcinosis (i.e., gout andCPPD). 11. A history of abnormal laboratory results >2.5×ULN indicativeof any significant medical disease, which in the opinion of theinvestigator, would preclude the subjects participation in the study.12. Any of the following abnormal laboratory results during screening:a. ALT and AST >2.5×ULN b. Hemaglobin <9 g/dL c. WBC <3500 cells/mm3. d.Lymphocyte count <1000 cells/mm³ e. Serum creatinine >1.5×ULN. 13. Ahistory of malignancy in the past ten years (<10 years), with theexception of resected basal cell carcinoma, squamous cell carcinoma ofthe skin, or resected cervical atypia or carcinoma in situ. 14.Significant hip pain, ipsilateral to the index knee that may interferewith assessments of index knee pain. 15. A known or clinically suspectedinfection with human immunodeficiency virus (HIV), or hepatitis C or Bviruses. 16. Participated within 3 months or will participateconcurrently in another investigational drug or vaccine study. 17. Ahistory of drug or alcohol dependence or abuse in the past 3 years 18. Afemale with reproductive capability who is unwilling to use birthcontrol for the duration of the study and/or intends to conceive within12 months of dosing. 19. Other serious, non-malignant, significant,acute or chronic medical or psychiatric illness that, in the judgment ofthe investigator, could compromise subject safety, limit the subject'sability to complete the study, and/or compromise the objectives of thestudy.

All subjects are monitored for AEs during the study. Assessments mayinclude monitoring of any or all of the following parameters: thesubject's clinical symptoms; laboratory, pathological, radiological, orsurgical findings; physical examination findings; or other appropriatetests and procedures. AEs that cause a subject to discontinue studyparticipation must be followed up until either the event resolves,stabilizes, or returns to baseline (if a baseline assessment isavailable).

This trial is entitled “Hydroxychloroquine/Atorvastatin in the Treatmentof Osteoarthritis (OA) of the Knee” and was registered onClinicalTrials.gov as NCT01645176. To date, 7 human medial compartmentknee OA patients have met inclusion criteria and been enrolled. Therewere no drop-outs of subjects who initiated combination HCQ+atorvastatintherapy in our trial. There were no serious adverse events in the trial.All 7 subjects have now completed 16 weeks of dosing, including allbaseline, in-life and follow-up examinations, tests andgadolinium-enhanced MRI imaging studies. As presented in FIG. 25, acombination of HCQ+atorvastatin (the combination termed “Arthrostatin”)reduced joint inflammation in humans with medial-compartment knee OA inthis 16-week open-label clinical trial. The MRI Synovitis Score wasmeasured by gadolinium-enhanced MRI scanning of the affected knee ineach subject at baseline and at the end of the 16-week in-lifeHCQ+atorvastatin treatment period, and represents the degree ofinflammation in the joint. Only candidate subjects with an abnormal MRIsynovitis score of 9-14, along with other clinical and inflammatorymarkers, were enrolled and treated. Subjects were treated with acombination of HCQ 600 mg by mouth each day and atorvastatin 40 mg bymouth each day for 16 weeks. The MRI Synovitis Scores were analyzed bytwo-way paired T test, which demonstrated that treatment with acombination of HCQ+atorvastatin statistically reduced the amount ofsynovitis (inflammation) in the affected knee joints (P=0.024) (FIG.25).

Further, in the 7 medial-compartment knee OA patients enrolled, acombination of HCQ+atorvastatin reduced the WOMAC Pain Score, WOMACFunction Score and WOMAC Combined Score in human with medial-compartmentknee OA patients in this 16-week open-label clinical trial. In thistrial, we also measured Western Ontario and McMaster UniversitiesArthritis Index (WOMAC) Pain, Functional and Combined Scores (seeMcConnell et al., The Western Ontario and McMaster UniversitiesOsteoarthritis Index (WOMAC): a review of its utility and measurementproperties. Arthritis Rheum2001; 45: 453-61. PMID:11642645). The WOMACPain, Function and Combined scores were analyzed by one-tailed T tests,which demonstrated that treatment with a combination of HCQ+atorvastatinstatistically reduced the WOMAC Pain Score at 16 weeks (P=0.035), WOMACFunction Score (P=0.005), and the WOMAC Combined Score (P=0.003) (FIG.25 B-D).

Thus, our 16-week open-label pilot trial of the combination ofHCQ+atorvastatin in humans with medial-compartment knee OA demonstratesthat this combination reduced synovitis (inflammation) in the affectedknee (P=0.024; FIG. 25 A), and resulted in improvements in the WOMACPain, Function and Combined Scores (FIG. 25 B-D). Together, these datasuggest that a combination of HCQ+atorvastatin provides meaningfulclinical benefit to, and reduced inflammation in, and thus may reduce OAdisease progression in humans.

Drugs that reduce inflammation may provide disease-slowing effectsincluding chondroprotection (e.g. reduction in the rate of cartilagebreakdown). Specifically, the combination of HCQ+atorvastatin may notonly reduce synovitis but that this reduction in synovitis will resultin a slowing of OA disease progression. This slowing of OA diseaseprogression in subsequent phase II and phase III trials will bedemonstrated by weight-bearing plain film X-rays of the affected kneethat demonstrate preservation of joint space (e.g. slowing of thenarrowing of the joint space in the medial compartment of the affectedknee) and/or that knee MRI scans will demonstrate preservation ofcartilage volume and/or integrity (and thus slowing of diseaseprogression).

New methods are being developed for measuring cartilage volume andintegrity, and these new methods will be used in subsequent phase II andphase III studies to demonstrate that HCQ+atorvastatin together protectagainst cartilage loss in human OA. An example of the methods foranalyzing joint space narrowing by plain X-ray in medial-compartmentknee OA are described in Brandt et al. (Arthritis and Rheumatism,52(7):2015-2025, PMID: 15986343), and the slowing of joint spacenarrowing is considered to demonstrate disease-slowing activity in OA. Asecond and more sensitive method to demonstrate chondroprotection is thedemonstration of preservation of cartilage volume on MRI scan, and anexample of methods of using MRI to demonstrate cartilage volumepreservation are described in Raynauld et al. (Ann Rheum Dis. 2009,68(6):938-47. PMID: 18653484).

Given the potent anti-inflammatory properties of DHCQ, DHCQ+atorvastatincould provide even more greater efficacy in reducing synovitis onGd-MRI, reducing WOMAC pain scores, and improving WOMAC function scoresin human OA.

Example 15 Use of DHCQ, or Combination Therapy with DHCQ+Atorvastatin,to Prevent Development of Osteoarthritis (OA)

Humans are screened for evidence of early OA or increased risk for thedevelopment of OA. Many factors can put humans in a preclinical OAdisease state including joint injury, joint surgery, degenerativemeniscal tears, degeneration of articular cartilage, anterior cruciateligament tears, collagen and other matrix protein defects, geneticpredisposition, and other factors. Humans in the process of developingOA or with features of early OA can be treated with DHCQ or with acombination of DHCQ+atorvastatin to prevent the development andprogression of OA. Further, humans at risk for OA or with early OA canbe further tested for the presence of inflammation in the involved jointto identify individuals most-likely to respond to treatment with DHCQ orwith DHCQ+atorvastatin. Testing for joint inflammation can be performedwith imaging markers, such as MRI with or without gadolinium contrast,or an ultrasound, to determine if one or more of the following abnormalimaging markers indicative of inflammation are present: synovialenhancement or proliferation, an effusion is present, and bone marrowedema. Molecular markers of inflammation can also be tested to identifyabnormal molecular inflammatory markers, including abnormal levels ofone or more of CRP, ESR and inflammatory cytokines. Finally, clinicalhistory and exam can be used to assess inflammation—including thepresence of abnormal clinical markers including an effusion on physicalexam or morning stiffness on history.

The dose of DHCQ can be about 400 mg/day (about 6.7 mg/kg/day), but canbe about 500 mg/day (about 8.3 mg/kg/day), or can be about 550 mg perday (9.16 mg/kg/day), or can be about 600 mg/day (about 10 mg/kg/day),or can be about 800 mg/day (about 13.3 mg/kg/day), or can be between100-1600 mg/day (about 1.6-26.67 mg/kg/day). The dose of atorvastatin isgenerally about 20 or about 40 mg/day (about 0.33-0.66 mg/kg/day), butcan be between about 5 and 80 mg/day (about 0.08-1.3 mg/kg/day). TheDHCQ, or DHCQ+atorvastatin, can be delivered in individual tablets orcapsules, or in a combined tablet or capsule that includes both drugs.

Examples of humans at high risk for development and with preclinical OA,and their treatment with DHCQ or combination therapy withDHCQ+atorvastatin, include:

(1) A 59 year old male with knee pain is diagnosed with osteoarthritisof the R knee (Kellgren-Lawrence, K-L, grade II). He is limited whenrunning and sitting for prolonged periods by the sensation of stiffnessor “gelling” in his knee. His R knee range of motion is intact and thereis no deformity of angulation of adduction moment on ambulation.Assessment is performed using the Western Ontario and McMasterUniversities (WOMAC) OA index for assessment of pain, function andstiffness of the knee joint as well as a score of 1-100 using a visualanalog score (VAS) for pain. The patient undergoes MRI with gadoliniumof the R knee which measures and reveals enhancement consistent withsynovitis which is assessed using a semi-quantitative scoring system.Based on the abnormal clinical and imaging markers, the patient isdetermined to be in the early-stages of OA and is therefore treated witha DHCQ 550 mg taken once daily as a combination capsule. Another MRI isrepeated at six months along with an evaluation to determine if there isa decrease in synovitis.

(2) 44 year old male amateur rugby player develops left knee pain andclicking with running. He is evaluated by X-ray which demonstrates K-Lgrade 1 changes and a knee MRI which reveals a posterior meniscal tearand he is scheduled for arthroscopic debridement. Blood tests reveal anelevated (abnormal) C-reactive protein (CRP) of 3.1. Beginning one monthbefore surgical debridement the patient is treated with DHCQ 200 mgdaily for 1 week, then 800 mg daily for 3 weeks, then 600 mg dailythereafter.

(3) 54 year old male presents with mild intermittent locking in his leftknee. X-ray reveals K-L grade 1 OA and ultrasound demonstrates adegenerative meniscal tear and moderate synovial enhancement consistentwith synovitis. The patient is offered arthroscopic meniscal debridementbut declines surgical intervention. He is prescribed DHCQ 600 mg.

(4) 28 year old male develops a fracture of his right ankle (tibialplafond) with appropriate reduction and casting. His X-rays do not showany features of OA. Given the 30% risk of significant radiographic OAwithin 2-4 years increasing to 74 percent by 11 years after fracture,the patient is monitored for evidence of joint inflammation byultrasound and MRI, and/or by molecular markers. Ultrasound detects(measures) a synovial effusion and synovitis, and as a result thepatient is determined to be at increased risk for progression to OA andis therefore started on DHCQ 300 mg twice daily (for a total dose of 600mg per day [10 mg/kg/day]).

(5) 49 year old male presents with intermittent pain in his left knee.X-ray reveals K-L grade 1 OA and MRI demonstrates a degenerativemeniscal tear and moderate synovial enhancement consistent withsynovitis. The patient is offered arthroscopic meniscal debridement butdeclines surgical intervention. Based on the clinical history and thepresence and measurement of abnormal clinical and imaging markers, he isdetermined to have early-stage OA. To treat and prevent the progressionof his early-stage OA, he is prescribed a combination of DHCQ 550 mgdaily+atorvastatin 40 mg daily.

Example 16 Use of DHCQ for Treatment of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a systemic inflammatory disorderwith protean manifestations. The involvement of endosomal toll-likerececptors (TLRs including TLR7 and TLR) have been strongly implicatedin disease pathogenesis (Rahman et al, 2008. NEJM. (9)929-939,PMID#18305268) and the therapeutic efficacy of hydroxychoroquine,presumably acting via endosomal inhibition, has been well established(Rahman et al, 2008. NEJM. (9)929-939, PMID#18305268). However, as SLEis a chronic disease, most patients require long term therapy with HCQand a reason for discontinuation is ocular deposition and potentialocular toxicity (Terahi et al, 2008. Semin Opthal. (3):201-208. PMID#.18432546).

Examples of humans at high risk for development, with preclinical, orwith established SLE, and their treatment with DHCQ, include:

(1) A 22 year old female presents with malar rash, arthralgia and isfound to have nephritis as evidenced by elevated serum creatinine, and aurinalysis demonstrating red blood cell casts. She is treated withprednisone, cyclophosphamide, and HCQ and after several monthsdemonstrates remission of all symptoms except mild arthralgia and returnof her malar rash with heavy sun exposure. Cyclophosphamide isdiscontinued and prednisone is tapered off. Given evidence for reducedrisk of relapse in SLE patients treated with ongoing HCQ she isinstructed to continue for life. Given the need for long term therapy,there is increased risk of retinal toxicity. To avoid retinal toxicity,the patient is switched from HCQ to DHCQ treatment and after 5 years ofaminoquinoline therapy ophthalmic exams for retinal toxicity areinitiated on an every 2-year schedule.

(2) A 34 year old female with a 12 year history of SLE is seen in followup. Initial presentation included rash, arthritis, and acuteglomerulonephritis which was treated with prednisone andcyclophosphamide which was then transitioned to azathioprine and HCQ.Flares have occurred on approximately six occasions each responsive tolow to moderate doses of prednisone. However, on a recent ophthalmologicevaluation using fundus autofluorescence (FAF), early changes consistentwith possible retinopathy are observed. Given the established efficacyof antimalarial therapy to prevent and decrease the severity of flaresof SLE, in the setting of early retinopathy, her treatment is changedfrom HCQ to DHCQ 550 mg daily. She continues on DHCQ 550 mg dailywithout progression of maculopathy on yearly opthalmic examination forthe subsequent 10 years. Furthermore she notes no increased rate orseverity of SLE flares over the same 10 years.

(3) A 28 year old female medical resident is diagnosed with SLE based onmalar rash, photosensitivity, arthritis, oral ulcers, and pleuritis. Sheis 52 kg and is treated with hydroxychloroquine 200 mg with only partialresponse to therapy. She is highly resistant to use corticosteroids dueto her small frame and low bone mass and she declines immunosuppressantsdue to the frequent contact with sick patients. She and the physicianare aware of her increased risk of retinal toxicity with a higher doseof hydroxychloroquine, thus she is prescribed DHCQ 300 mg twice per day(600 mg total per day) with resolution of all SLE signs and symptoms.Opthamologic screening at 5 and 10 years of treatment with DHCQdemonstrates no evidence of retinal toxicity, and after 10 years oftreatment with DHCQ ophthalmic monitoring is performed every 2 years tomonitor for retinal toxicity, and over an additional 10 years of therapyno retinal toxicity is observed.

Example 17 Use of DHCQ for Treatment of Non-Alcoholic Fatty LiverDisease (NASH) in Humans

Non-alcoholic fatty liver disease (NALFD) is a common conditioncharacterized by fat deposition in the liver. Patients with NAFLD are atsignificant risk for development of non-alcoholic steatohepatitis, aninflammatory liver disease dependent on TLR4 activation by endotoxin inthe gastrointestinal tract. Given the observed ability ofhydroxychloroquine to abrogate LPS mediated TLR4 activation,prescription of hydroxychloroquine is considered. However, the patientis a known type 2 diabetic, a condition commonly co-existing withNAFLD/NASH, and this has resulted in early diabetic retinopathy. AsNASH/NAFLD is a chronic disease, long term therapy with HCQ would berequired putting the patient at risk for ocular deposition and potentialocular toxicity.

Examples of humans at high risk for development of, with preclinical, orwith established NASH, and their treatment with DHCQ, include:

(1) A 59 year old a man is seen on routine follow up for longstandingType 2 diabetes. He is noted to have elevated AST and ALT to twice theupper limit of normal. He is asymptomatic and reports no hepatoxicmedications. Abdominal ultrasounds is consistent with fatty infiltrationof the liver. Based on the clinical presentation and abnormalinflammatory and imaging markers, the patient is determined to be athigh risk for progression to NASH. Therapy with hydroxychloroquine isconsidered but in an effort to decrease risk of retinal toxicity, thepatient is treated with DHCQ 550 mg per day, which prevents the patientfrom progressing to NASH, and after 10 years of DHCQ therapy the patientis screened for retinal toxicity and none is found and ophthalmicscreening is continued on an every 5 year basis.

(2) A 53 year old man is seen for polyuria (frequent urination). He isfound to have a fasting glucose of 390 mg/dL and hemoglobin A1C of 8.5mg/dL>It is noted that his hepatic enzymes (ALT and AST) are nearlythree times the upper limit of normal and high sensitivity CRP (hsCRP)is elevated at 2.1. He is otherwise asymptomatic and reports nohepatoxic medications. Abdominal ultrasounds is consistent with fattyinfiltration of the liver and liver biopsy reveal hepatic steatosis withclusters of macrophages. Therapy with insulin and metformin is initiatedwith control of blood sugar and at 6 months hemoglobin A1c is reduced to7.5 mg/dL. However, AST and ALT are still nearly three times the upperlimit of normal and hsCRP is 1.9. Based on the measured abnormalclinical, abnormal metabolic, and abnormal imaging markers, the patientis determined to have NASH. HCQ is considered but in an effort todecrease risk of retinal toxicity, the patient is treated with DHCQ 600mg/day, which prevents the patient from progressing results innormalization of AST and ALT and a reduction in hsCRP to 0.9 mg/dL.Notably, hemoglobin A1c is reduced to 6.1 without further titration ofprimary hypoglycemic therapy. After 10 years of DHCQ therapy the patientis screened for retinal toxicity and none is found, and the patientsubsequently screened every other year by ophthalmic examination forretinal toxicity.

Example 18 Treatment of the Metabolic Disease Non-AlcoholicSteatohepatitis (NASH) with DHCQ

A 49 year old man has elevated liver enzymes with an alaninetransaminase (ALT) level of 59 IU/L and an aspartate transaminase (AST)level of 55 IU/L. Ultrasound of the liver will be consistent with fattyinfiltration and serologic tests will be negative for hepatitis B or Cvirus and he will deny use of alcohol. The patient is found to have animpaired fasting glucose level of greater than 120 and elevatedtriglycerides greater than 320 mg/dL. He undergoes a liver biopsy thatdemonstrates steatosis ballooning, degeneration of hepatocytes, as wellas mixed portal inflammation but no fibrosis. Based on these abnormalfindings and markers he is diagnosed with NAFLD and early NASH, andbased on this diagnosis he is prescribed DHCQ 600 mg per day. Serumsamples are collected at baseline and after 2 months of treatment toevaluate for levels of alanine aminotransferase (ALT; also known asserum glutamic pyruvate transaminase [SGPT]) and aspartate transaminase(AST; also known as serum glutamic oxaloacetic transaminase [SGOT])alanine aminotransferase (ALT) as well as a panel of multiplexcytokines. Reductions in the ALT, AST and/or cytokines indicate apositive response to treatment.

Example 19 Treatment of the Metabolic and Inflammatory Diseases:Treatment of Type II Diabetes and the Metabolic Syndrome with DHCQ

Examples of humans at high risk for development, with preclinical, orwith established type II diabetes and/or metabolic syndrome, and theirtreatment with DHCQ, include:

(1) A 42 year old man with history of obesity (BMI 31) found to have afasting glucose of 106 mg/dL, an LDL level of 135, and a triglyceridelevel of 220. Evaluations for secondary causes of hyperglycemia arenegative and based on the abnormal metabolic markers he is determined tobe at increased risk for development of an inflammatory disease ordisease associated with inflammation. Based on this increased risk, heis treated with atorvastatin 40 mg and DHCQ 400 mg daily.

(2) A 48 year old man with history of obesity (BMI 30) is found to havea fasting glucose of 121 mg/dL, an LDL level of 135, and a triglyceridelevel of 220. High sensitivity CRP (hsCRP) is elevated at 1.9. He istreated for 4 months with atorvastatin 40 mg with reduction in LDL to105 and hsCRP to 1.7. He is then started DHCQ 400 mg daily with furtherreduction in LDL to 95 and hsCRP to 0.9. In addition, fasting glucoselevels fall to 101 without progression to type 2 diabetes over a periodof 5 years.

(3) A 53 year old man with a history of hypertension and previousmyocardial infarction is noted to have an LDL cholesterol level of 140mg/dL and a high sensitivity CRP (hsCRP) of 1.6 mg/L. He is treated withatorvastatin with a decrease in his LDL to 115 mg/dl and a fall in hishsCRP to 1.2. He is subsequently treated with DHCQ 600 mg daily,clinically does well with further reduction in LDL to 99 mg/dl and hsCRPto 0.8. After 10 years of therapy he is screened for and has no evidenceof retinal toxicity.

Example 20 Use of DHCQ for Treatment of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a systemic inflammatory disorderwith protean manifestations. The involvement of endosomal toll-likerececptors (TLRs including TLR7 and TLR) have been strongly implicatedin disease pathogesis (Rahman et al, 2008. NEJM. (9)929-939,PMID#18305268) and the therapeutic efficacy of hydroxychoroquine,presumably acting via endosomal inhibition, has been well established(Rahman et al, 2008. NEJM. (9)929-939, PMID#18305268). However, as SLEis a chronic disease, most patients require long term therapy with HCQand a major reason for discontinuation is ocular deposition andpotential ocular toxicity (Terahi et al, 2008. Semin Opthal.(3):201-208. PMID#. 18432546).

Examples of humans at high risk for development, with preclinical, orwith established SLE, and their treatment with DHCQ, include:

(1) A 22 year old female presents with malar rash, arthralgia and isfound to have nephrotic syndrome as evidenced by elevated serumcreatinine and a urinalysis demonstrating red blood cell casts. She istreated with prednisone, cyclophosphamide, and HCQ and after severalmonths demonstrated remission of all symptoms except mild arthralgia andreturn of her malar rash with heavy sun exposure. Cyclophosphamide isdiscontinued and prednisone is tapered off. Given evidence for reducedrisk of relapse in SLE patients treated with ongoing HCQ she isinstructed to continue for life. Given the need for long term therapy,there is increased risk of retinal toxicity. To avoid retinal toxicity,the patient is switched from HCQ to DHCQ treatment.

(2) A 34 year old female with a 12 year history of SLE is seen in followup. Initial presentation was for rash, arthritis, and acuteglomerulonephritis which was treated with prednisone andcyclophosphamide which was then transitioned to azathioprine. Flareshave occurred on approximately six occasions each responsive to low tomoderate doses of prednisone. However, on a recent ophthalmologicevaluation using fundus autofluorescence (FAF), early changes consistentwith possible hydroxycholoroquine retinopathy. Given the establishedefficacy of antimalarial therapy to prevent and/or decrease flares ofSLE but in the setting of early hydroxychloqoruine maculopathy, she istreated with DHCQ 400 mg daily without progression of maculopathy onyearly examination for the subsequent 10 years. Furthermore she note noincreased rate or severity of SLE flares over the same 10 years.

(3) A 28 year old female medical resident is diagnosed with SLE based onmalar rash, photosensitivity, arthritis, oral ulcers, and pleuritis. Sheis 52 kg and is treated with hydroxychloroquine 200 mg with only partialresponse to therapy. She is highly resistant to use corticosteroids dueto her small frame and low bone mass and she declines immunosuppressantsdue to the frequent contact with sick patients. She and the physicianare aware of her increased risk of retinal toxicity with a higher doseof hydroxychloroquine, thus she is prescribed DHCQ 600 mg day withresolution of all SLE signs and symptoms. Yearly opthamologic screeningreveals no evidence of retinopathy at 5 and 10 years of therapy.

(4) A 30 year old female is tired, develops a malar rash, and is foundto have an anti-Sm antibody titer of 1:320. She is at-risk for thedevelopment of SLE, and is treated with DHCQ 550 mg/day, her symptomsimprove, and after 5 years of therapy retinal monitoring is started onan every-other year basis and no retinal toxicity is observed.

Example 21 Treatment of the Metabolic and Inflammatory Disease Type IIDiabetes with DHCQ

Examples of humans at high risk for development, with preclinical, orwith established type II diabetes, and their treatment with DHCQ,include:

(1) A 42 year old man with history of obesity (BMI 31) is found to havea fasting glucose of 106 mg/dL, an LDL level of 135, and a triglyceridelevel of 220. Evaluations for secondary causes of hyperglycemia arenegative and he is treated with atorvastatin 40 mg and DHCQ 400 mgdaily.

(2) A 53 year old man with a history of hypertension and previousmyocardial infarction is noted to have an LDL cholesterol level of 140mg/dL. He is treated with atorvastatin with a decrease in his LDL to 115mg/d. He is subsequently treated with DHCQ 600 mg daily, clinically doeswell, and after 10 years of therapy is screened for and has no evidenceof retinal toxicity.

Example 22 Treatment of the Chronic Immune Activation and MetabolicAbnormalities in HIV Infection with DHCQ

A 38 year old man with a 9 year history of HIV disease, treated with atriple drug regimen of anti-retroviral therapy has an undetectably viralload (<10,000 copies/ml) and CD4 T cell count of 490. He feels well andhas had no opportunistic infections. He is noted to have impairedfasting glucose level of 109 mg/dL and elevated triglycerides at 299mg/dL. High sensitivity C-reactive protein (hsCRP) level is 5.8 mg/L. Acoronary CT scan reveal significant calcification of the coronaryarteries with an Agatston score of 124 but an exercise stress testreveals no inducible cardiac ischemia. Based on these abnormalinflammatory and abnormal metabolic markers, he is prescribed DHCQ 600mg daily, clinically does well, and after 10 years of therapy isscreened for and has no evidence of retinal toxicity.

Example 23 Treatment of Atherosclerosis with DHCQ

Examples of humans at high risk for development, with preclinical, orwith established atherosclerosis, and their treatment with DHCQ,include:

(1) A 59 year old man with a history of hypertension is evaluated forexertional chest pain. Exercise stress imaging reveals a reversibleregion of ischemia in the lateral wall of the heart and he is taken tocardiac catheterization which reveals diffuse lesions of 40-60% stenosisin the left main and left anterior descending coronary arteries. He istreated with DHCQ 500 mg per day and he does not have further myocardialinfarction.

(2) A 48 year old man with a no active medical problems and nomedications is seen for a first episode of acute chest pain lasting 1hour and diagnosed with a non-ST elevation myocardial infarction. He hasa family history of premature coronary artery disease. Cardiac stresstest reveals no focal areas of defect on by electrocardiogram (ECG) ornuclear perfusion imaging. His LDL is 161 mg/dL. He is treated andreleased with new medication regimen including daily aspirin 81 mg andatorvastatin 40 mg. At follow up in 8 weeks he is symptom free with anLDL of 90 but is noted to have an ALT of 71 and AST of 66, both of whichwere previously normal. A dose reduction of atorvastatin to 20 mgresults in minimal chance in ALT/AST but with a rise in LDL to 140, wellabove goal for LDL for known coronary disease. He is prescribed DHCQ 400mg daily to take in addition to atorvastatin 20 mg daily with a fall inLDL to 110 mg/dL and normalization of his AST/ALT. His atorvastatin isincreased back to 40 mg with a reduction in LDL to 80 mg/dL without risein AST/ALT. Therapy is continued for over 10 years without stable LDL,AST/ALT as well as normal retinal exam at 5, 10, and 20 years oftherapy.

Example 24 Use of Desethylhydroxychloroquine (DHCQ), or CombinationDHCQ+Atorvastatin, Therapy to Prevent Development of and to Reduce theSeverity of Rheumatoid Arthritis (RA)

Humans are screened for evidence of early RA or for being at high riskfor the development of RA. Findings that suggest an individual human hasearly RA include one or more of the following: presence of one or moreswollen joints, the presence of anti-CCP or rheumatoid factorantibodies, evidence of synovial enhancement on MRI scan or ultrasound,and markers including elevations in autoantibodies and cytokinesdemonstrated to provide predictive utility for the subsequentdevelopment of RA (as described in Sokolove et al, PLoS One. 2012;7(5):e35296, PMID: 22662108). Factors that place an asymptomaticindividual, or individual with monoarthritis, at increased risk fordevelopment of RA include one or more of the following: a family historyof RA (particularly in a first-degree relative), increased anti-CCPand/or rheumatoid factor antibodies, and a genetic profile withincreased susceptibility to RA, and/or one or more joints exhibitingsynovitis (joint swelling and inflammation).

Further, humans at high risk for RA or with early RA can be tested forthe presence of inflammation in the involved joint to identifyindividuals most-likely to respond to treatment with DHCQ or treatmentwith a combination of DHCQ+atorvastatin. Testing for joint inflammationcan be performed with imaging markers, such as MRI with or withoutgadolinium contrast, or an ultrasound, to determine if one or more ofthe following features indicative of inflammation are present: synovialenhancement or proliferation, an effusion is present, and bone marrowedema. Molecular markers of inflammation can also be tested for,including one or more of CRP, ESR and inflammatory cytokines. Finally,clinical history and exam are used to assess inflammation—including thepresence of synovitis on physical examination, an effusion on physicalexam, or morning stiffness >1 hour on history.

Individual humans with preclinical or early RA, particularly whenevidence of inflammation is found using imaging, molecular or clinicalmarker, can be treated with DHCQ, or a combination of DHCQ+atorvastatin,to prevent the progression of pre-clinical or early RA. The dose of DHCQis generally at least about 400 mg/day, but can be between about100-1600 mg/day, or between about 550 and 1000 mg/day. The DHCQ can bedelivered in individual tablets or capsules, or the combinedDHCQ+atorvastatin delivered as a combined tablet or capsule thatincludes both drugs.

All publications and patent documents cited herein are incorporated byreference in their entirety. To the extent the material incorporated byreference contradicts or is inconsistent with the present specification,the present specification will supersede any such material.

1. A pharmaceutical composition comprising: a unit dose of 25 to 3000 madesethylhydroxychloroquine or salt and/or ester thereof effective intreating inflammation with decreased retinal toxicity in combinationwith an effective dose of one or more statins; and a pharmaceuticallyacceptable excipient. 2-39. (canceled)
 40. The composition of claim 1,wherein the statin is selected from the group consisting ofatorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin, and simvastatin.
 41. The composition of claim40, wherein the statin is present at a dose of from 1 to 250 mg.
 42. Thecomposition of claim 41, wherein the statin is present as aconcentration of from 5 to 60 mg.
 43. The composition of claim 1,wherein the dose of desethylhydroxychloroquine is about 100-600 mg. 44.A method of treating an inflammatory disease comprising: administeringto an individual in need thereof a pharmaceutical composition comprisinga unit dose of 25 to 3000 mg desethylhydroxychloroquine, or a saltand/or ester thereof effective in treating inflammation with decreasedretinal toxicity in combination with an effective dose of one or morestatins; and a pharmaceutically acceptable excipient.
 45. The method ofclaim 44, wherein a daily amount of desethylhydroxychloroquineadministered to the individual for the first 1 to 16 weeks is higherthan the daily amount of desethylhydroxychloroquine administeredsubsequently.
 46. The method of claim 44, wherein the statin is selectedfrom the group consisting of atorvastatin, fluvastatin, lovastatin,mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.47. The method of claim 46, wherein the statin is present at a dose offrom 1 to 250 mg.
 47. The method of claim 47, wherein the statin ispresent as a concentration of from 5 to 60 mg.
 48. The method of claim44, wherein the dose of desethylhydroxychloroquine is about 100-600 mg.49. The method of claim 44, wherein the inflammatory disease is adegenerative disease selected from the group consisting ofosteoarthritis, Alzheimer's disease, and macular degeneration.
 50. Themethod of claim 44, wherein the inflammatory disease is a metabolicdisease selected from the group consisting of type II diabetes,atherosclerosis and non-alcoholic steatohepatitis.
 51. The method ofclaim 44, wherein after 10 years of said administration, the individualis substantially without symptoms of retinal toxicity.
 52. The method ofclaim 44, wherein there is reduced screening for retinal toxicity. 53.The method of claim 52, wherein screening starts after 10 years of saidadministration.
 54. The method of claim 44, wherein there is noscreening for retinal toxicity.